The cell wall is the battleground where plants and pathogens attempt to prevail by implementing contrasting wall-reinforcing and wall-weakening strategies. When pathogens start degrading the plant cell wall components, plants are capable of perceiving the loss of wall integrity and subsequently activate the defense signaling pathways. Pathogens try to escape the plant defenses and sometimes take advantage of the host cell wall metabolism to facilitate their entry into the tissue. These dynamic processes vary according to the lifestyle of the pathogen and the type of plant pathogen interaction. While necrotrophy involves a strong and diffused molecular warfare that may provoke extended lesions of the tissue, during biotrophy the battle involves a weaker cell wall degradation mainly localized at the point of penetration and at the level of the feeding apparatus. Perception of cell wall damage as well as the pathogen- and host-induced cell wall remodeling occurs in both cases. The damage of specific cell wall polysaccharides during infection may be perceived by receptors as THE1, ER and WAK1. Plants may also rely on the recognition of CWDEs by LRR-RLPs receptors, as RBPG1 and LeEIX1-2. Cell wall fragments may be released during infection and sensed as damage signals. Analysis of cell wall mutants has shed light on the relationship between cell wall remodeling and plant response to pathogens. The expression of endogenous and microbial CWDEs and their inhibitors is also a valuable approach for studying the dynamics of the cell wall during plant– pathogen interactions as well as a strategy to improve plant protection.

#### **ACKNOWLEDGMENTS**

We would like to apologize to those people whose relevant publications could not be cited because of space constraints. This research was supported by the European Research Council (ERC Advanced Grant, 233083).

#### **REFERENCES**

pectin degradation in tabasco pepper. *J. Plant Physiol.* 163, 488–496. doi: 10.1016/j.jplph.2005.06.022

*Arabidopsis* are required for resistance to the necrotrophic fungus *Plectosphaerella cucumerina. Plant J.* 43, 165–180. doi: 10.1111/j.1365-313X.2005. 02440.x

**Conflict of Interest Statement:** The Review Editor, Dr Ferrari, declares that, despite being affiliated to the same institution as authors Dr Daniela Bellincampi and Dr Felice Cervone, the review process was handled objectively. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 21 March 2014; paper pending published: 10 April 2014; accepted: 06 May 2014; published online: 28 May 2014.*

*Citation: Bellincampi D, Cervone F and Lionetti V (2014) Plant cell wall dynamics and wall-related susceptibility in plant–pathogen interactions. Front. Plant Sci. 5:228. doi: 10.3389/fpls.2014.00228*

*This article was submitted to Plant–Microbe Interaction, a section of the journal Frontiers in Plant Science.*

*Copyright © 2014 Bellincampi, Cervone and Lionetti. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited andthatthe original publication inthis journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# The role of the cell wall in plant immunity

# *Frederikke G. Malinovsky1\*, Jonatan U. Fangel 2 andWilliam G. T.Willats 2*

*1 DNRF Center DynaMo and Copenhagen Plant Science Center, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Copenhagen, Denmark*

*2 Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Copenhagen, Denmark*

#### *Edited by:*

*Vincenzo Lionetti, Sapienza Università di Roma, Italy*

#### *Reviewed by:*

*Thorsten Hamann, Norwegian University of Science and Technology, Norway Kian Hématy, Institut National de la Recherche Agronomique, France*

#### *\*Correspondence:*

*Frederikke G. Malinovsky, DNRF Center DynaMo and Copenhagen Plant Science Center, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg Centret, Copenhagen, Denmark e-mail<EMAIL_ADDRESS>
The battle between plants and microbes is evolutionarily ancient, highly complex, and often co-dependent. A primary challenge for microbes is to breach the physical barrier of host cell walls whilst avoiding detection by the plant's immune receptors. While some receptors sense conserved microbial features, others monitor physical changes caused by an infection attempt. Detection of microbes leads to activation of appropriate defense responses that then challenge the attack. Plant cell walls are formidable and dynamic barriers. They are constructed primarily of complex carbohydrates joined by numerous distinct connection types, and are subject to extensive post-synthetic modification to suit prevailing local requirements. Multiple changes can be triggered in cell walls in response to microbial attack. Some of these are well described, but many remain obscure. The study of the myriad of subtle processes underlying cell wall modification poses special challenges for plant glycobiology. In this review we describe the major molecular and cellular mechanisms that underlie the roles of cell walls in plant defense against pathogen attack. In so doing, we also highlight some of the challenges inherent in studying these interactions, and briefly describe the analytical potential of molecular probes used in conjunction with carbohydrate microarray technology.

**Keywords: plant cell wall, defense, PTI, PAMP, DAMP, callose, chitin, immunity**

# **INTRODUCTION**

The dynamic interplay between pathogen and plant host is the product of millions of years of co-evolution. This struggle is often described in terms of an "arms race," a fitting term considering the investment required and the significance for both sides. The goal for the plant is to keep healthy and fertile, but for pathogens the strategy varies. Biotrophic and hemibiotrophic microorganisms rely on keeping their host alive and unaware of their presence, at least until later stages of infection. In contrast, necrotrophs live and feed on dead and dying cells and can therefore use a more forceful attack strategy (Davidsson et al., 2013). The frontline of the plant defense system consists of physical and chemical barriers such as the cell wall, waxes, hairs, antimicrobial enzymes, and secondary metabolites. If these obstacles are overcome, the pathogen is still confronted by elaborate surveillance systems in which molecular sentinels operate to activate resistance responses (Jones and Dangl, 2006).

Molecular components that serve essential functions for the fitness or survival of microbes are often highly conserved. For plants, detection of such microbial fingerprints also referred to as pathogen-associated molecular patterns (PAMPs), is a warning of impending attack (Medzhitov and Janeway, 1997). Consequently, a key aspect of plant innate immunity is the ability to recognize PAMPs such as bacterial flagellin, lipopolysaccharides, peptidoglycans, and fungal chitin (Boller and Felix, 2009). In addition to sensing PAMPs, the ability to sense a compromised "self" by detecting damage-associated molecular patterns (DAMPs) such as released plant cell wall fragments is a central part of plant defense (Boller and Felix, 2009). Both PAMP- and DAMPrecognition activates PAMP-triggered immunity (PTI) which in

general prevents microbial colonization. To escape detection and PTI induction, a common strategy among adapted microorganisms is to secrete a range of effector proteins that can modulate PTI components (Jones and Dangl, 2006). The stealth afforded by the microbial effectors can in turn be counteracted in the plant by an intracellular surveillance system consisting of an array of nucleotide-binding leucine-rich-repeat proteins that seek to detect the presence of such effector proteins, and enable induction of effector-triggered immunity (ETI). ETI is often associated with a localized cell death termed the hypersensitive response which functions to restrict spread of this more progressive stage of microbial attack (Jones and Dangl, 2006; Dodds and Rathjen, 2010). Hence the important feature of PTI is the ability to sense *infectious-self* and *non-self*, whereas for ETI it is the ability to sense microbe-mediated modifications inferred on points of vulnerability in the host. By guarding these weak points or even setting up decoys to confuse invaders, ETI is an efficient safety net for more progressed infections (van der Hoorn and Jones, 2004; Jones and Dangl, 2006).

Pathogen-associated molecular pattern perception is mediated by ligand-binding surface-exposed transmembrane patternrecognition receptors (PRRs) of either the receptor-like kinase (RLK) or receptor-like proteins (RLPs) families. Both types of modular proteins are single-pass transmembrane proteins with extracellular domains, but where RLKs have an intracellular kinase domain; RLPs lack this cytosolic signaling domain (Monaghan and Zipfel, 2012). The archetypical bacterial PRRs are elongation factor Tu (EF-Tu) receptor (EFR), a leucine-rich repeat RLK (LRR-RLK) that recognizes the abundant cytoplasmic protein EF-Tu; and the related flagellin sensing 2 (FLS2) that recognizes flagellin, the principal component of bacterial flagella. These two PAMPs are often characterized by their minimal requirement peptide epitopes, elf18 and flg22 (Gomez-Gomez et al., 1999; Kunze et al., 2004; Boller and Felix, 2009). Sensing of the elf18 peptide appears to be restricted to the *Brassicaceae* family, while in rice recognition of EF-Tu, occurs via the EFa50 region (Furukawa et al., 2013). Interestingly, transgenic expression of *Arabidopsis* (*Arabidopsis thaliana*) EFR in *Solanaceae* species that cannot perceive EF-Tu is sufficient to confer resistance to a broad range of phytopathogenic bacteria, suggesting high conservation of the responses downstream of PAMP recognition (Lacombe et al., 2010). PTI induction leads to a series of early and late responses. The early responses occur within minutes to hours, and consist of rapid ion fluxes across the plasma membrane, an oxidative burst, activation of mitogen-activated protein kinases (MAPKs) and calcium-dependent protein kinases (CDPKs), and induction of defense-related genes. Deposition of callose, inhibition of seedling growth and PAMP induced resistance are later responses that occur within days (Boller and Felix, 2009).

#### **RECOGNITION OF CHITIN: AN EVOLUTIONARY ARMS RACE**

A good example of the intricate evolutionary arms race between a pathogen and its plant host concerns the polysaccharide chitin (**Figure 1**). Chitin, a homopolymer of β-(1→4)-linked *N*-acetylglucosamine (GlcNAc) units, is the major structural component of fungal cell walls, and is also a main constituent of insect exoskeletons, crustacean shells, and the eggs and gut linings of nematodes (Bueter et al., 2013; Hadwiger, 2013). Chitin is an obvious PAMP-candidate and an ideal point of attack during plant defense responses since glucosamine polymers are not found in plants. It is therefore not surprising that an evolutionarily conserved strategy toward fungi and insects in plants is based on secreting chitinases – hydrolytic enzymes, which

can break down microbial chitin polymers (**Figure 1**; Hadwiger, 2013). However, as usual, countermeasures have also evolved, the biotrophic fungal pathogen *Cladosporium fulvum* combats the action of chitinases by secreting the apoplastic effector Avr4, a chitin-binding lectin that functions to protect the integrity of the fungal cell wall against chitinases (**Figure 1**; van den Burg et al., 2006). Heterologous expression of Avr4 in *Arabidopsis* or tomato camouflages the chitin thereby increasing the virulence of several fungal pathogens (van Esse et al., 2007). That is unless the host harbors Cf-4, an extracellular membrane-anchored LRR protein that mediates Avr4 perception (**Figure 1**) and activates the hypersensitive response (Thomas et al., 1997; Takken et al., 1999). In addition to this forceful degradation strategy, chitin is also recognized as a PAMP in rice (*Oryza sativa*) by a dual recognition system consisting of the lysine motif (LysM)–RLK CHITIN ELICITOR RECEPTOR KINASE-1 OsCERK1 and the LysM-RLP CHITIN OLIGOSAC-CHARIDE ELICITOR-BINDING PROTEIN OsCEBiP (Shimizu et al., 2010). In *Arabidopsis* there is no contribution to signaling from the major chitin-oligosaccharide binding CEBiP, and AtCERK1 seems to act alone as the chitin PRR (**Figure 1**; Iizasa et al., 2010; Petutschnig et al., 2010; Shinya et al., 2012). The biological activity of chitin oligomers depends on their size, as the highest PAMP-activity is found for heptamers and octamers. Higher oligomeric chitin fragments like octamers can bind two or more AtCERK1 and this ligand-induced dimerization activates the receptor (Liu et al., 2012). To avoid chitin-induced PTI, *C. fulvum* also secretes the evolutionally conserved LysMcontaining effector extracellular protein 6 (Ecp6) during infection. Ecp6 is a scavenger of chitin fragments released by chitinases and out-competes chitin-PRR binding to avoid fungal detection (**Figure 1**; Bolton et al., 2008; de Jonge et al., 2010; Sanchez-Vallet et al., 2013). Whether Ecp6 is recognized in plants is still

**FIGURE 1 |The "arms race" between** *Cladosporium fulvum* **and plant hosts.** As a general protective measure against fungal infections plants respond to infection attempts by secreting chitinases into the apoplastic space. The tomato leaf mould fungus *C. fulvum* can enter its host through stomatal openings, and grows as extracellular hyphae. To shield against the action of these chitin-degrading enzymes the fungus camouflages its chitin-containing cell walls by cloaking them with the chitin-binding effector Avr4 (van den Burg et al., 2006; Hadwiger, 2013). The presence of Avr4 can be recognized by the Tomato RLP Cf-4, leading to induction of the hypersensitive response (Thomas et al., 1997; Takken et al., 1999). Chitin oligomers released by chitinases are recognized as PAMPs, in *Arabidopsis* by the RLK CERK1 (Iizasa et al., 2010; Petutschnig et al., 2010), and in rice by both OsCERK1 and the RLP OsCEBiP (Shimizu et al., 2010). To escape chitin-induced PTI *C. fulvum* can secrete Ecp6 an effector that functions as a chitin-scavenger removing the chitin oligomers released by the chitinases (de Jonge et al., 2010; Kombrink and Thomma, 2013; Sanchez-Vallet et al., 2013).

unknown. In all cases, this example demonstrates the continuous battle between pathogen and host and how one party's armor is continuously being evolutionarily countered by the opponent.

### **PTI SIGNALING**

A vital feature of plant innate immunity is the early recognition of potential pathogens via perception of PAMPs and DAMPs. This recognition is mediated by designated surface-localized PRRs and sets in train a range of early and late responses that ensure a specialized and appropriate response is transmitted by a shared downstream pathway. Some of these steps are already mapped whereas others are yet to be discovered.

After ligand recognition, several PRRs rapidly form complexes with the regulatory RLK BRI1-ASSOCIATED KINASE1 (BAK1; **Figure 2**; Monaghan and Zipfel, 2012). Indeed, complex formation between the co-receptor and the flagellin receptor FLS2 occurs within seconds of flg22 treatment (Schulze et al., 2010). BAK1 was believed to function solely as a signal enhancer rather than being involved in ligand-binding. However, it has recently been demonstrated that BAK1 acts as a co-receptor in FLS2 mediated flg22 recognition by interacting with the bound ligand (Sun et al., 2013). BAK1 belongs to the somatic embryogenesis-related kinases; a small family of five LRR-RLKs (Heese et al., 2007; Roux et al., 2011). In addition to FLS2, BAK1 has also been shown to interact with EFR and the DAMP receptors PEPR1 and PEPR2 (Krol et al., 2010; Yamaguchi et al., 2010). Apart from its association with PRRs, BAK1 is also involved in the perception of brassinosteroids through its interaction with the RLK BRASSI-NOSTEROID INSENSITIVE 1 (BRI1; Li et al., 2002; Nam and Li, 2002). PAMP-binding and complex formation leads to auto- and trans-phosphorylation between PRR and co-receptor, as well as the plasma membrane localized receptor-like cytoplasmic kinase BOTRYTIS-INDUCED KINASE 1 (BIK1) and related PBS1-LIKE (PBL) kinases (Lu et al., 2010; Zhang et al., 2010). BIK1 plays a central role in conveying signals from several PRRs including EFR, FLS2, CERK1, and PEPR1/PEPR2. PAMP/DAMP induced phosphorylation of BIK1 prompts dissociation from the PRR complex which may permit BIK1 to move toward and activate downstream signaling targets (Lu et al., 2010; Zhang et al., 2010; Liu et al., 2013).

# **CONVEYING THE SIGNAL INTO TRANSCRIPTIONAL REPROGRAMMING**

Calcium ions serve as important second messengers in eukaryotes. Both abiotic and biotic stress responses lead to rapid transient fluctuations in the concentration of intracellular calcium ions (**Figure 2**). The low basal cytosolic concentration allows rapid spatial and temporal changes in the calcium flux. Such calcium signatures are sensed by stimulus-specific Ca2+-binding sensors that can mediate the downstream signal transduction. Deciphering the calcium signatures enables the affected cells to respond appropriately to diverse stimuli (DeFalco et al., 2010). During PTI, CDPKs function as Ca2+-sensors that are required to obtain complete transcriptional reprogramming (**Figure 2**). The four functionally redundant *Arabidopsis* CDPKs, CPK4, CPK5, CPK6, and CPK11 define a sub-clade requiredfor flg22 induced PTI responses (Boudsocq et al., 2010). The transcriptional changes effected by the CDPKs are mostly independent of the MAPK pathway (see below), but the combination of CDPKs and MAPKs is needed to activate

**FIGURE 2 | A current model of AtFLS2 signaling.** Upon flg22 binding, a complex between FLS2, BIK1, BAK1 (and other SERKs) is formed. Complex formation triggers multiple rapid phosphorylation events resulting in BIK1 release. The signal transduction downstream of

ligand perception includes a Ca2+ burst, activation CDPKs and AtRbohD required for the ROS burst, and induction MAPK cascades. Activation of CDPKs and MAPKs is required for full induction of defense genes.

at least four early transcriptional regulatory programs induced by flg22 (Boudsocq et al., 2010; Wurzinger et al., 2011).

An oxidative burst of reactive oxygen species (ROS) such as ·O2 −, ·OH, and H2O2 is one of the early measurable events in PTI (**Figure 2**). In *Arabidopsis*, the ROS burst is facilitated by the RESPIRATORY BURST OXIDASE HOMOLOGUE D (RbohD), an NADPH oxidase (Torres et al., 2002). The activity of RbohD is dependent on calcium and phosphorylation (Ogasawara et al., 2008; Kimura et al., 2012), and interestingly RbohD is phosphorylated by CDPK5 (Dubiella et al., 2013). In potato (*Solanum tuberosum*) StRbohB is phosphorylated by the plasma membranelocalized potato StCDPK5, and expression of constitutively active StCDPK5 in *Nicotiana benthamiana* leads to induction of ROS (Kobayashi et al., 2007).

Like the CDPKs, MAPK cascades also function to transmit signals within the cell via differential phosphorylation. In such cascades MAP kinase-kinase-kinases (MAPKKKs) phosphorylate MAP kinase-kinases (MAPKKs), which in turn phosphorylate MAP-kinases (**Figure 2**; Rasmussen et al., 2012). The *Arabidopsis* MAP kinases MPK3, MPK4, MPK6 as well as the recently documented MPK11 are all activated by PAMP treatment (Rasmussen et al., 2012). They designate at least two different pathways: one that triggers MPK4 activation by a module consisting of the two MAPKKs MKK1 and MKK2, and MEKK1; and another that leads to MPK3/MPK6 activation, via MKK4 and MKK5, and possibly MEKK1 (Rasmussen et al., 2012). Activation of MAPK cascades is, together with CDPK activation, vital for obtaining the transcriptional reprogramming needed to mount a full PTI response (**Figure 2**; Boudsocq et al., 2010). Even though some genes require both MAPK and CDPK activation to be induced there seems to be a branching of PTI signals on some level (Boudsocq et al., 2010; Xu et al., 2014). Loss of the flg22-induced ROS in *Arabidopsis rbohD* does not affect MPK3 and MPK6 activation, and activation of these MAP kinases is also not required for ROS burst (Xu et al., 2014).

# **FATE OF ACTIVATED RECEPTORS**

Ubiquitination is a key signal for endosomal sorting of membrane proteins (Raiborg and Stenmark, 2009). Upon ligand perception FLS2 is being ubiquitinated by two PLANT U-BOX (PUB) E3 ligases PUB12 and PUB13 (Lu et al., 2011). This may well be part of a strategy to terminate the signal initiated by PAMP recognition. FLS2 is endocytosed and targetedfor endocytic degradation approximately 30–60 min post-elicitation, and the activated FLS2 travels through early, late and multi-vesicular endosomes toward its vacuolar destination (Robatzek et al., 2006; Chinchilla et al., 2007; Beck et al., 2012b; Smith et al., 2014). The removal of ligand-bound receptors de-sensitizes the system after initial PAMP-treatment. After two hours the de-sensitized cells begin to be re-sensitized through *de novo* synthesis mediated replenishment of the receptor, preparing them for a new round of ligand perception (Smith et al., 2014). FLS2 has recently been shown to interact with two subunits of ENDOSOMAL SORT-ING COMPLEXES REQUIRED FOR TRANSPORT-I (ESCRT-I; Spallek et al., 2013). ESCRT complexes are responsible for identifying ubiquitinated vesicular cargoes, sorting them for degradation, and depositing them in intraluminal vesicles to prevent their

recycling (Raiborg and Stenmark, 2009). Loss of the E3 ligases or ESCRT-I components affects flg22-triggered immune reactions underlining the importance of correct endosomal trafficking of FLS2 in sustaining a sufficient level of immunity (Lu et al., 2011; Spallek et al., 2013). In addition removal of activated receptors, trafficking might also play an integrated part in signaling transduction as part of the signaling responses may happen from endosomal compartments (Geldner and Robatzek, 2008; Beck et al., 2012a).

# **LATE PTI RESPONSES**

The final consequence of PTI is the induction of resistance responses that will prevent microbial colonization. Other late responses are the inhibition of seedling growth inhibition occurring within days of continuous PAMP treatment, and deposition of callose about 16 h after PAMP treatment (Boller and Felix, 2009). Callose is found during attempted fungal infections in cell wall fortifications termed papillae, structures that are assembled at fungal penetration sites (Underwood, 2012). The linear β-(1→3)-D-Glc*p* homo-polymer is produced by callose synthases; among these is the *Arabidopsis* POWDERY MILDEW RESISTANT 4 (PMR4), the predominant synthase responsible for defense-induced callose deposition (Vogel and Somerville, 2000; Nishimura et al., 2003; Ham et al., 2007; Chen and Kim, 2009). All the callose deposited in response to flg22, and most of the chitosan-induced depositions are synthesized by PMR4 (Luna et al., 2011). In addition to differences in their dependency on PMR4, callose depositions induced by these two PAMPs also differ in their requirements for other signaling components as only the flg22-induced callose depends on RbohD (Luna et al., 2011). Callose depositions like the papillae are an important feature of immunity, and are thought to reinforce the cell wall at fungal penetration sites to impede infections (Underwood, 2012). In agreement with this, overexpression of PMR4 in *Arabidopsis* results in enlarged callose deposits, and gain of penetration resistance against both virulent and non-adapted powdery mildew strains (Ellinger et al., 2013; Naumann et al., 2013). Absence of callose depositions also leads to susceptibility toward the non-adapted hemibiotrophic bacteria *Pseudomonas syringae* pv. *phaseolicola* (Ham et al., 2007). Surprisingly the *pmr4* mutant was identified by its increased powdery mildew resistance, a phenotype that is dependent on the defense phytohormone salicylic acid (Vogel and Somerville, 2000; Nishimura et al., 2003; Huibers et al., 2013). These observations are counterintuitive both with respect to the pattern of callose deposition during defense induction, and the recent evidence from overexpression lines, supporting a role for callose in the plants defensive strategy (Vogel and Somerville, 2000; Ellinger et al., 2013; Naumann et al., 2013). One explanation for such a discrepancy could be that PMR4 is guarded by a resistance gene. Accordingly, loss of PMR4 might be perceived as a breach on the plants defensive fences and thus set about induction of ETI via elevation of salicylic acid.

# **THE ROLE OF PLANT CELL WALLS IN MICROBIAL INTERACTIONS**

Plant cell walls not only provide structure to the plant body but also act as barriers against biotic and abiotic stresses. The cell wall, sometimes covered with a cuticle, is usually the first obstacle encountered by pathogens, and to penetrate this barrier microbes have evolved an arsenal of wall degrading enzymes which are key virulence factors (**Figure 3**; Nuhse, 2012; Davidsson et al., 2013).

Most plant cell walls are based on a co-extensive load-bearing network of cellulose micro-fibrils cross-linked with hemicelluloses (**Figure 3**; Fry, 2004; Scheller and Ulvskov, 2010; Pauly et al., 2013). In the primary walls of the growing parts of plants, this network is embedded in a matrix of pectic polysaccharides. In the secondary cell walls of mature, non-growing tissues pectin

is less abundant but walls are reinforced with lignin (Endler and Persson, 2011). Although most cell walls are based on these main components they differ considerably in their fine structures and three dimensional architectures. This heterogeneity is reflected in the diversity of strategies that pathogens have evolved to breach them, including the secretion of numerous glycosyl hydrolases (Annis and Goodwin, 1997). In response to an attack, plants may deposit certain reinforcing polymers, notably callose, phenolic complexes, and employ toxic compounds (Huckelhoven, 2007). However, these physical and chemical responses are only one part of the cell walls role in defense

**FIGURE 3 |The plant cell wall. (A)** The primary cell wall is constructed of a web of cellulose micro-fibrils, hemicellulose polysaccharides, and the hetero-polysaccharide pectin (Endler and Persson, 2011). **(B)** Necotrophic pathogens devour their hosts by degrading the plant cell wall, and secreting necrosis-inducing proteins and toxins (Davidsson et al., 2013). The host entry step of such microbes is thought to happen mainly via secretion of cell wall degrading enzymes such as cellulases, pectinases, and hemicellulases (Choquer et al., 2007; Davidsson et al., 2013). Xylanase, degrades the linear backbone of the predominant hemicellulose xylan into xylose residues (Belien et al., 2006; Scheller and Ulvskov, 2010). Pectinases such as polygalacturonases (PGs) and pectate lyases (PLs) degrades the pectic homogalacturonan (HGA) backbone. The HGA in newly synthesized pectin is methyl-esterified, which protects it from degradation by pectinases. De-esterification of HGA by the action of microbial pectin methyl esterases (PMEs) enables access PGs and PLs (Annis and Goodwin, 1997; Wolf et al., 2009; Lionetti et al., 2012). Endo-PGs cleave un-esterified regions while concomitantly releasing the oligogalacturonides (OGAs), oligomeric fragments of the HGA backbone (Annis and Goodwin, 1997; Ferrari et al., 2013). The wall-associated kinases (WAKs) are DAMP sensors that monitor the integrity of pectin by sensing the presence of OGAs (Ferrari et al., 2013). Plants can counter the cocktail of cell wall degrading enzymes by producing proteinaceous inhibitors such as the xylanase inhibitors and PG inhibitor proteins (PGIPs; Belien et al., 2006).

and the extensive ability of pathogens to degrade cell wall component has a cost. Disturbance of cell wall integrity (which may also cause deformation of adjacent portion of the plasma membrane) and the release of degradation fragments are monitored by plants and changed cell wall status is an important trigger for defense mechanisms (Hematy et al., 2009). Despite the undoubted importance of cell walls in plant defense there are many aspects that are poorly understood. For example, most work has focused on a limited set of cell wall polymers (notably callose, extensins, and lignin) and the potential roles of many other cell wall components are obscure. Another largely unexplored aspect of cell walls and defense is the evolution of and co-evolution of defense strategies (Sørensen et al., 2010). Discussed below are the major cell wall components in the context of pathogenic responses to them, and the prospects for advancing our understanding using emerging microarray technologies.

#### **CELLULOSE**

Tightly packed crystalline cellulose micro-fibrils serve a primary structural function in the cell wall (Endler and Persson, 2011; Nuhse, 2012). The fibrils are composed of hydrogen-bonded β- (1→4)-D-Glc*p* chains, and have a condensed nature that makes them generally resistant to degradation via glycosyl hydrolases. However, recent work has highlighted the role of another class of oxidative enzymes, the lytic polysaccharide monoxygenases that have the capacity to degrade recalcitrant crystalline cell wall components, including cellulose (Vaaje-Kolstad et al., 2010; Hemsworth et al., 2014). Cellulose micro-fibrils are synthesized by large multimeric complexes containing cellulose synthase catalytic subunits (CESAs; Endler and Persson, 2011). It is generally considered that in *Arabidopsis*, and probably other angiosperms, there are two CESA subfamilies with distinct roles. Whilst CESAs 1, 3, and 6 are responsible for the formation of cellulose in primary walls, CESAs 4, 7, and 8 synthesize cellulose in secondary walls (Endler and Persson, 2011). However, recent work has demonstrated that some functional cross over exists across these groups *in vitro* although it is not clear if these new CESA pairings are significant *in planta* (Carroll et al., 2012).

As in many cases where cell wall integrity is perturbed, compensatory mechanisms have evolved to limit the effects of disturbance to cellulose structure or deposition. Cellulose deficient mutants typically exhibit increased lignification (Cano-Delgado et al., 2003; Hamann, 2012). Interestingly, these changes appear to have effects beyond the purely structural. Such mutants also display enhanced defense responses (Hamann, 2012) as mutants in the primary wall *CESA3* are more resistant toward powdery mildew (Ellis and Turner, 2001; Cano-Delgado et al., 2003). Additionally defects in the secondary wall CESAs; CESA4, CESA7, and CESA8, also lead to elevated resistance against broad-host necrotrophic pathogens like the fungus *Plectosphaerella cucumerina* and the soil-borne bacterium *Ralstonia solanacearum* (Hernandez-Blanco et al., 2007). Consistent with the genetic evidence, similar results have also been described for treatment with the cellulose biosynthesis inhibitor isoxaben (Hamann et al., 2009; Hamann, 2012). In *Arabidopsis*, isoxaben-mediated wall damage and lignin production is induced via an RbohD-dependent mechanism, and fine-tuned through a jasmonic-acid-dependent negative feedback loop (Hamann et al., 2009; Denness et al., 2011). The defense associated cellulose deprivation phenotypes suggest that the cell wall damage triggers defense responses, and suggest the presence of a tight cell wall integrity monitoring system. The RLK THESEUS1 (THE1) seems to serve a role in this as it has been implicated in the response to cell wall damage. The *the1* mutant was identified as a suppressor of the *cesA6* null allele *procuste1-1.* Mutations in *THE1* reduce the ectopic lignification of several cellulose deficient mutants (Hematy et al., 2007).

#### **HEMICELLULOSES**

Hemicelluloses are a diverse group of polysaccharides usually characterized by having a β-(1→4)-linked backbone of mannose, glucose, or xylose. The central role of hemicelluloses is to fortify the cell wall by interaction with cellulose and sometimes lignin (Scheller and Ulvskov, 2010; Endler and Persson, 2011). Xylans are the predominant hemicelluloses in secondary plant cell walls. The common characteristic of xylans is a backbone of repeating β-(1→4)-D-Xyl*p* residues most often substituted by Ara*f* and Glc*p*A (Scheller and Ulvskov, 2010). The arabinosyl residues can in addition contain ferulic acid groups esterified to the O-5 position of the carboxyl group (Smith and Hartley, 1983). These ferulate esters can be oxidatively cross-linked to lignin possibly incorporating xylan into the lignin complex to advance strengthening of the network (Tanner and Morrison, 1983; Mcneil et al., 1984; Harris and Stone, 2009). Some phytopathogenic microbes secrete xylanases, which are enzymes that can degrade the linear xylan backbone into xylose units (Belien et al., 2006). Hemicellulose breakdown by xylanases weakens the cell wall and enables the microbe to breach it (Belien et al., 2006). Fungi of the *Trichoderma* spp. produce the ETHYLENE-INDUCING XYLANASE (EIX) that is recognized as a PAMP (Furman-Matarasso et al., 1999; Belien et al., 2006). In tomato (*Lycopersicum esculentum*), EIX is sensed by the cell wall-derived RLPs LeEix1 and LeEix2 (**Figure 3**; Ron and Avni, 2004).

In addition to feruloylation cell wall glycans can also be subjected to methylation and acetylation. REDUCED WALL ACETYLATION 2 (RWA2) has been shown to be responsible for the acetylation of numerous pectic and non-pectic polymers in *Arabidopsis*, and *rwa2* knock-out mutants have (Mcneil et al., 1984) increased resistance toward *Botrytis cinerea* (Manabe et al., 2011). Furthermore de-acetylation of xyloglucan and pectin in transgenic plants exhibit increased accessibility to degrading enzymes which could be part of a defense strategy to release oligosaccharides which can act as defense elicitors (Pogorelko et al., 2013).

#### **PECTIN**

The complex hetero-polysaccharide pectin is a major cell wall matrix component that is abundant in primary cell walls. It is composed of a series of structurally distinct domains acting as backbones or side chains to the pectic polysaccharides (Wolf et al., 2009). Back bones are comprised either of contiguous α-(1→4)-D-Gal*p*A residues (homogalacturonan or HGA) or repeating dimers of α-(1→4)-D-Gal*p*A-α-(1→2)-L-Rha*p* residues (rhamogalacturonan). Within the pectin complex especially HGA appears to have special significance in the context of defense responses. HGA can be methyl esterified at C6 and acetylated at C2–C3 and regulation of these substitutions enable plants to fine-tune the functionality of HGA to suit prevailing local requirements. For example non-esterified HGA carrying negatively charged free carboxyl groups may be subject to cross-linking via calcium leading to the formation of gels that are required for cell adhesion and other structural roles (Cabrera et al., 2008).

Some of the first enzymes that phytopathogenic fungi secrete during infections are endo-polygalacturonases (PGs) which cleave HGA (**Figure 3**), thereby degrading cell wall integrity and aiding pathogen access (Annis and Goodwin, 1997). Moreover, HGA degradation releases oligogalacturonide (OGA) fragments from the backbone, which act as potent defense response elicitors (Galletti et al., 2009). The WALL-ASSOCIATED KINASES (WAKs) are thought to be DAMP sensors that monitor the integrity of pectin by detecting the presence of OGAs with a degree of polymerization between 10 and 15 (Ferrari et al., 2013). In the context of degradation by microbial enzymes the level of HGA methyl esterification is critically important. Endo-PGs and pectate lyases (PLs) preferentially cleave non-esterified HGA and these enzymes frequently act in concert with pectin methyl esterases (PMEs) that de-methyl esterify HGA to create endo-PG and PL cleavage sites (**Figure 3**). Moreover, another class of microbial enzymes – the pectin lyases act preferentially on highly methyl esterified HGA.

Homogalacturonan and the microbial enzymes that degrade it are good examples of how the high complexity of cell wall components is countered by sets of highly specific microbial enzymes. Nevertheless, the role of pectin in plant defense cannot only be viewed in straightforward terms of polysaccharide integrity versus enzymatic breakdown. An example is provided by the elevated activity of certain endogenous *Arabidopsis* PMEs in response to infection with the necrotrophic fungus *Alternaria brassicicola*, the hemibiotrophic bacterial pathogen *P. syringae pv. maculicola* ES4326, or by treatment with PAMPs (Bethke et al., 2014). The increase in PME activity in such infected plants leads to a concomitant decrease in methyl esterification of HGA, which doubtlessly renders the HGA more susceptible toward endo-PGs (Bethke et al.,2014). It seems counterintuitive to increase the activity of an endogenous wall degrading enzyme during a pathogen attack as this could aid microbial entry. One explanation is that the enhanced production of OGAs – and thereby an associated potential activation of DAMP signaling – is worth the price of a decrease in HGA integrity. It is also important to consider the broader picture of the 66 PMEs in *Arabidopsis* in terms of redundancy and spatiotemporal regulation. For example, in the case of *A. brassicicola* it is worth noting that the observed increase in PME activity occurred rather late after pathogen challenge. With this in mind it may be the case that the purpose of enhanced PME activity is localized HGA de-methyl esterification and the concomitant formation of gel structures to bolster damaged walls (Bethke et al., 2014).

Another noteworthy insight into the roles of pectin in defense is provided by the loss of function mutants of the endogenous plant enzyme POWDERY MILDEW RESISTANT 6 MUTANT (PMR6; Vogel et al., 2002)*.* PMR6 has similarity to PLs, and loss of function leads to enhanced levels of intact pectin and but also lower levels of potential OGA release*.* One explanation for the enhanced resistance to powdery mildew in *pmr6* is that increased levels of inaccessible HGA *per se* provide more protection. But, the fact that *pmr6* only exhibits this elevated resistance against certain powdery mildew species (*Erysiphe cichoracearum* and *E. orontii*) implies a less generic and more subtle effect. For example, it is possible that alterations in *pmr6* cell wall composition make these mutants poor hosts specifically for *Erysiphe* spp*.* It certainly seems highly unlikely that a plant gene would have evolved to serve the needs of a pathogen, and the pleotropic effects observed in *pmr6* are consistent with a role as a susceptibility factor. It is plausible that *pmr6* resistance is a specialized form of disease resistance, possibly based on the loss of a host susceptibility gene required by the pathogen for growth and development (Vogel et al., 2002).

# **LIGNIN**

Lignin is a phenolic polymer mainly deposited in secondary cell wall during the last stages of cell differentiation. It displaces the aqueous phase of the cell wall, encasing cellulose and matrix polysaccharides and providing enhanced mechanical strength and a water-impermeable barrier (Albersheim et al., 2011). Lignin is also required for reinforcing vascular cells that transport water under negative pressure as a result of transpiration. The importance of lignin in these tissues is demonstrated by vascular collapse in lignin deficient plants (Piquemal et al., 1998; Jones et al., 2001). Lignin is built from monolignols, with up to three different types in higher plants which appear to be incorporated into the lignin polymer in a non-predictable fashion (Martone et al., 2009; Vanholme et al., 2010). This apparently random pattern of synthesis may be significant in relation to microbial enzymes which have typically evolved to break polymers with structurally consistent cleavage sites (Sarkar et al., 2009).

Lignin has multiple roles in plant defense and lignin, or ligninlike phenolic polymers are often rapidly deposited in response to both biotic and abiotic stresses (Sattler and Funnell-Harris, 2013). Lignin not only acts as physical barrier to pathogen invasion, but the phenylpropanoid pathway responsible for lignin biosynthesis may also be recruited for defense purposes. For example, this pathway underpins the synthesis of other phenolic compounds including phytoalexins, stilbenes, coumarins, and flavonoids – some of which have been implicated in plant defense (Weiergang et al., 1996; Dicko et al., 2005; Lozovaya et al., 2007). Indeed, there is evidence that in some plants, salicylic acid which is known to be a key component of some defense pathways, may also be produced by the phenylpropanoid pathway (Ruuhola et al., 2003; Pan et al., 2006). Understanding the role of lignin in plant defense has received considerable attention in the context of engineering plants for use as bioenergy feedstocks. Lignin contributes substantially to the recalcitrance of cell walls to deconstruction into fermentable sugars and for this reason reducing lignin content has become an important goal for feedstock biotechnology research. However, since lignin is also required for plant defense there is probably a lower limit for lignin reduction beyond which

plant would become unacceptably vulnerable to pathogens (Sattler and Funnell-Harris, 2013). The roles of lignin in defense and the importance of this for bioenergy feedstocks are reviewed in (Sattler and Funnell-Harris, 2013).

### **INHIBITORS OF MICROBIAL WALL DEGRADING ENZYMES**

Often, an infection involves a multifaceted strategy. In the case of *B. cinerea* the host entry step is thought to happen mainly via secretion of cell wall degrading enzymes such as a PMEs, endo-PGs, and endo-β-1,4-xylanase often encoded by multi-genic families (Choquer et al., 2007). Cellulases, pectinases, and hemicellulases are also secreted from bacteria like the *Pectobacterium* and *Dickeya* genera necrotrophic soft rot enterobacteria (Davidsson et al., 2013). At first glance, plants would seem defenseless against such savage enzyme cocktails, however, they can counter by producing proteinaceous inhibitors of cell wall degrading enzymes such as the xylanase inhibitors and PG inhibitor proteins (PGIPs; Belien et al.,2006). The cunning strategy of making PGIPs is not to inhibit pectin degradation entirely, but to shift it toward producing longer OGAs that can be sensed as DAMPs (Federici et al., 2006).

# **THE CHALLENGES OF MONITORING CELL WALL RESPONSES TO PATHOGENS**

Even from a concise overview such as provided above, it is clear that analyzing the subtle, complex and often rapid changes that affect cell walls in response to pathogens is a formidable technical challenge. In contrast to proteins and nucleotides, the polysaccharides from which cell walls are primarily made cannot readily be sequenced, synthesized, or expressed. Although cell walls are usually physically tough, they are also typically highly plastic and can undergo rapid modifications in response to specific local conditions, even within cell wall micro-domains. The analytical difficulties are compounded by the sheer complexity and multilayered nature of host/pathogen interactions, and the differing nature of these interactions depending on developmental stage, organ, or tissue. Conventional biochemical techniques for carbohydrate analysis, for example methylation analysis and ion exchange chromatography, are powerful but low throughput and usually require large amounts of material. They also involve the complete or partial destruction of cell walls into their component parts – which inevitably means that information about three dimensional cell wall architectures is lost. Molecular probes, for example monoclonal antibodies (mAbs) and carbohydrate binding modules (CBMs) have been invaluable for advancing our understanding of plant cell walls *per se*, and have immense potential for providing insight into host pathogen interactions at the cellular and subcellular levels. There is an extensive repertoire of probes directed against plant cell wall components (Lee et al., 2011), but this is not matched by probes for pathogens and their associated effectors. For example, our understanding of the precise role of chitosan (the de-acetylated form of chitin) during pathogenesis is hampered by a lack of suitable probes (Sorlier et al., 2003; Hoell et al., 2010; Baker et al., 2011).

One technique that appears to offer considerable promise for plant/pathogen interaction research is based on carbohydrate microarrays (Moller et al., 2007). This technique combines the specificity of mAbs and CBMs with the multiplexed analysis capacity of robotically produced microarrays, and simultaneously provides information about the relative abundance of multiple epitopes present in large sample sets (Tyler et al., 2014). This approach has been extensively used for cell wall analysis in numerous contexts (Tronchet et al., 2010; Sørensen et al., 2011; Fangel et al., 2012; Moore et al., 2014; Zhang et al., 2014), but has only been used in a very limited way for plant defense research (Nguema-Ona et al.,2013). Typically, at least 20 mAbs or CBMs are used in a single analysis, and several 100 samples can be processed in 2 days. Analysis starts with the preparation of cell wall material (although native plant material can also be used) which is then treated with a series of solvents to release cell wall components. For example, extraction with 1,2-diaminocyclohexanetetraacetic acid followed by sodium hydroxide is expected to release pectins and hemicellulose respectively. These extractions are then printed onto a suitable surface such as nitrocellulose membrane or modified glass slides using a microarray robot, and the resultant arrays probed with mAbs and/or CBMs and then quantified. The fact that relatively small amounts of cell wall material are needed (0.5– 10 mg) and the ability to rapidly analyze large numbers of samples are attractive attributes in the context of plant/pathogen interaction research. Currently, the analysis of cell walls in the context of plant defense is often limited to polymers with well characterized roles, for example extensins and callose. The described technology can potentially provide the means to greatly increase the scope of cell wall analysis, both in terms of the number of cell wall components analyzed and the number of variables in an experimental set up. Our initial experiments indicate that glycan arrays are powerful tools for monitoring the progression of cell wall changes during defense responses and are well suited to the parallel analysis of, for example, multiple mutants, elicitors, or conditions. Furthermore we anticipate that analysis of cell wall glycomes will be integrated with transcriptomic analysis of defense related genes using the same material. Moreover, although the technique has mostly been applied to carbohydrate analysis, the same approach could be applied to investigate proteins and their conjugates of relevance to pathogenic interactions. So long as analytes can be extracted and immobilized, and providing that probes are available the scope of analysis can be extended.

# **CONCLUDING REMARKS**

Our current knowledge of the interactions between plants and pathogens at the plant cell wall level, and how this affects downstream signaling is still limited. To obtain more insight we need to expand our understanding of the full repertoire of cell wall modifications that occurs during microbial interactions. Carbohydrate microarrays could be used to monitor the complex interplay between microbes and plants, and may reveal additional, perhaps more subtle, cell wall modifications to those documented so far. This technique enables rapid and multiplexed analysis of multiple changes in wall composition and could be used as a tool to provide new insight into the dynamic nature of host/pathogen interactions.

# **ACKNOWLEDGMENTS**

We thank Dr. Milena Roux and Dr. Jacqueline Monaghan for critically reading and commenting on the manuscript.

#### **REFERENCES**

synthesis of the endogenous flagellin-receptor FLAGELLIN-SENSING2. *Plant Physiol.* 164, 440–454. doi: 10.1104/pp.113.229179

Zhang, J., Li, W., Xiang, T., Liu, Z., Laluk, K., Ding, X., et al. (2010). Receptor-like cytoplasmic kinases integrate signaling from multiple plant immune receptors and are targeted by a *Pseudomonas syringae* effector. *Cell Host Microbe* 7, 290–301. doi: 10.1016/j.chom.2010.03.007

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 14 February 2014; accepted: 14 April 2014; published online: 06 May 2014.*

*Citation: Malinovsky FG, Fangel JU and Willats WGT (2014) The role of the cell wall in plant immunity. Front. Plant Sci. 5:178. doi: 10.3389/fpls.2014.00178*

*This article was submitted to Plant-Microbe Interaction, a section of the journal Frontiers in Plant Science.*

*Copyright © 2014 Malinovsky, Fangel and Willats. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited andthatthe original publication inthis journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Overexpression of a citrus NDR1 ortholog increases disease resistance in *Arabidopsis*

# *Hua Lu1\*, Chong Zhang1, Ute Albrecht 2, Rena Shimizu1, GuanfengWang1 and Kim D. Bowman2*

*1 Department of Biological Sciences, University of Maryland Baltimore County, Baltimore, MD, USA 2 United States Horticultural Research Laboratory, Agricultural Research Service, United States Department of Agriculture, Fort Pierce, FL, USA*

#### *Edited by:*

*Vincenzo Lionetti, "Sapienza" Università di Roma, Italy*

#### *Reviewed by:*

*Brad Day, Michigan State University, USA Robin K. Cameron, McMaster University, Canada*

# *\*Correspondence:*

*Hua Lu, Department of Biological Sciences, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250, USA e-mail<EMAIL_ADDRESS>
Emerging devastating diseases, such as Huanglongbing (HLB) and citrus canker, have caused tremendous losses to the citrus industry worldwide. Genetic engineering is a powerful approach that could allow us to increase citrus resistance against these diseases. The key to the success of this approach relies on a thorough understanding of defense mechanisms of citrus. Studies of *Arabidopsis* and other plants have provided a framework for us to better understand defense mechanisms of citrus. Salicylic acid (SA) is a key signaling molecule involved in basal defense and resistance (R) gene-mediated defense against broad-spectrum pathogens. The *Arabidopsis* gene *NDR1* (NON-RACE-SPECIFIC DISEASE RESISTANCE 1) is a positive regulator of SA accumulation and is specifically required for signaling mediated by a subset of R genes upon recognition of their cognate pathogen effectors. Our bioinformatic analysis identified an ortholog of *NDR1* from citrus, *CsNDR1*. Overexpression of *CsNDR1* complemented susceptibility conferred by the *Arabidopsis ndr1-1* mutant to *Pseudomonas syringae* strains and also led to enhanced resistance to an oomycete pathogen *Hyaloperonospora arabidopsidis*. Such heightened resistance is associated with increased SA production and expression of the defense marker gene *PATHOGENESIS RELATED 1* (*PR1*). In addition, we found that expression of *PR1* and accumulation of SA were induced to modest levels in citrus infected with *Candidatus* Liberibacter asiaticus, the bacterial pathogen associated with HLB disease. Thus, our data suggest that *CsNDR1* is a functional ortholog of *Arabidopsis NDR1*. Since *Ca.* L. asiaticus infection only activates modest levels of defense responses in citrus, we propose that genetically increasing SA/NDR1-mediated pathways could potentially lead to enhanced resistance against HLB, citrus canker, and other destructive diseases challenging global citrus production.

**Keywords:** *Pseudomonas syringae***, salicylic acid, citrus canker, Huanglongbing, greening disease,** *Candidatus* **Liberibacter asiaticus, genetic engineering**

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# **INTRODUCTION**

Huanglongbing (HLB; also called citrus greening disease), citrus canker, and other emerging diseases have imposed serious threats to the citrus industry worldwide (Bove,2006; Gottwald,2007). Citrus canker is caused by the gram negative bacterium *Xanthomonas axonopodis* pv. *citri*. Wind and rain facilitate the dispersal of the pathogen and spreading of the disease. More than 16 million citrus trees have been destroyed in Florida in an effort to restrict the disease (Gaskalla, 2006). HLB is even more devastating than citrus canker since it is highly contagious and lethal to afflicted plants (Bove, 2006; Brlansky and Rogers, 2007; Gottwald, 2007). The parasitic bacterium *Candidatus* Liberibacter that lives in the phloem tissue of a citrus tree is believed to be the associating agent of HLB. The disease is transmitted by a small insect vector of the family Psyllidae. The growth of psyllids cannot yet be reliably controlled by conventional insecticide application (Ichinose et al., 2010). Without successful measures to control the causal pathogen and its transmission vector, HLB is endemic to a variety of citrus species and related plants. Therefore, it is imperative to develop strategies to contain and eventually eradicate HLB and other diseases challenging the production of citrus worldwide.

Successful manipulation of citrus disease resistance relies on a thorough understanding of defense mechanisms of the plant. Although not well understood yet in citrus, defense mechanisms are well studied in other plants, in particular the model plant *Arabidopsis thaliana*, and have been shown to be relatively conserved among plants (Nishimura and Dangl, 2010). Therefore, prior knowledge of defense mechanisms from other plants should help us to better understand citrus defense and ultimately develop effective strategies to combat devastating diseases challenging the citrus industry.

Plant defense can be preformed or induced. The preformed defense includes some existing physical structures and chemical compounds produced by plants before infection that can restrict pathogen invasion. The induced defense can be activated upon pathogen invasion, involving sophisticated surveillance systems to recognize general elicitors from pathogens and subsequently to activate basal defense. Much stronger defense can be induced when host resistance (R) proteins specifically recognize their cognate pathogen effectors; thus such defense is also termed as R protein-mediated defense. The largest class of R proteins is represented by a family of proteins that have two conserved domains, nucleotide binding site (NBS) and leucine-rich repeat (LRR; Martin et al., 2003; McDowell and Simon, 2006). This class of R proteins can be further divided into two groups according to the N-terminal sequence, coiled-coil (CC)–NBS–LRR and Tollinterleukin-1 receptor (TIR)–NBS–LRR. Some CC–NBS–LRR type proteins are found to signal through NON-RACE-SPECIFIC DISEASE RESISTANCE 1 (NDR1). For instance, an *ndr1* mutation compromises resistance conferred by the CC–NBS–LRR proteins RPS2, RPM1, or RPS5 to *Pseudomonas syringae* expressing the avirulence effectors *avrRpt2*, *avrB* and *avrRpm1*, or *avrPph3*, respectively (Century et al., 1995; Aarts et al., 1998). On the other hand, some TIR–NBS–LRR type proteins functionally require ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1). For instance, an *eds1* mutant is immune-compromised to *P. syringae avrRps4*, resistance to which is conferred by the TIR–NBS–LRR protein RPS4 but not by TIR–NBS–LRR type R proteins (Aarts et al., 1998; Falk et al., 1999). These observations suggest a general rule that these two subgroups of R proteins can activate distinct downstream signaling pathways. However, exceptions to this rule also exist for some other NBS–LRR R proteins (McDowell et al., 2000; Bittner-Eddy and Beynon, 2001; Xiao et al., 2001).

The small phenolic molecule salicylic acid (SA) plays a key role in signaling both basal and R protein-mediated defense (Hammond-Kosack and Jones, 1996; Tsuda et al., 2008) and is involved in resistance against diverse pathogens and in response to various stress conditions (Malamy et al., 1990; Rassmussen et al., 1991; Sharma and Davis, 1997; Tsuda et al., 2008). While increased SA accumulation and/or signaling lead to enhanced disease resistance, disrupting these processes by gene mutations or transgene expression result in compromised defense against pathogens (Durrant and Dong, 2004; Lu, 2009). Genes involved in SA-mediated defense can affect SA biosynthesis, accumulation, and/or signaling (Lu, 2009). For instance, *SA INDUCTION-DEFICIENT 2*/*EDS16*, encodes isochorismate synthase contributing to the majority of SA biosynthesis (Wildermuth et al., 2001). Both *NDR1* and *EDS1* are known to act upstream of SA to regulate SA accumulation (Falk et al., 1999; Shapiro and Zhang, 2001). Downstream of SA signaling, *NONEXPRESSOR OF PR GENES 1* (*NPR1*) acts as a signal transducer that regulates systemic acquired resistance, a longlasting defense against broad-spectrum pathogens at the whole plant level (Cao et al., 1997; Ryals et al., 1997; Shah et al., 1997; Dong, 2004). Overexpression of *NDR1*, *EDS1*, *NPR1*, or several other SA regulators confers enhanced disease resistance to a range of pathogens in *Arabidopsis* and/or in other plants (Chern et al., 2001; Fitzgerald et al., 2004; Lin et al., 2004; Makandar et al., 2006; Malnoy et al., 2007; Pegadaraju et al., 2007; Sandhu et al., 2009; Gao et al., 2010). Therefore, manipulation of SA-mediated defense has the potential to introduce broad-spectrum disease resistance in plants.

*NDR1* encodes a glycosyl-phosphatidyl inositol-anchored plasma membrane protein that belongs to a large protein family (Dormann et al., 1995; Varet et al., 2002; Coppinger et al., 2004; Zheng et al., 2004). A recent study implicates the function of *NDR1* in mediating plasma membrane-cell wall adhesion (Knepper et al., 2011). *NDR1-*like genes widely exist in different plants (Lee et al., 2006; Chong et al., 2008; Cacas et al., 2011). Besides *NDR1*, some *Arabidopsis* homologs of *NDR1* were shown to be highly induced by pathogen infection and/or to confer enhanced disease resistance to *P. syringae* when overexpressed (Varet et al., 2002, 2003; Coppinger et al., 2004; Zheng et al., 2004). Thus *NDR1* and some members in the family are critical components of plant defense.

In this study, we report the isolation and characterization of a functional ortholog of *NDR1* in citrus, named *CsNDR1*. We found that overexpression of *CsNDR1* complements the susceptibility of *Arabidopsis ndr1-1* mutant to *P. syringae avrRpt2* and further confers enhanced disease resistance to *P. syringae avr-Rps4*, which normally is not affected by the endogenous *NDR1*. Overexpression of *CsNDR1* also led to increased resistance to the oomycete pathogen *Hyaloperonospora arabidopsidis* (*Hpa*) isolate Noco2. *CsNDR1*-induced disease resistance is associated with increased SA accumulation and expression of the defense marker gene *PATHOGENESIS RELATED 1* (*PR1*) in the transgenic *Arabidopsis* plants. In addition, we found that citrus infected with *Candidatus* Liberibacter asiaticus, a pathogen associated with the HLB disease, expressed modestly increased *CsNDR1* and SA levels, compared with mock-treated plants. We propose that genetic engineering to enhance SA/NDR1 signaling pathway in citrus could potentially enhance its resistance to HLB, citrus canker, and other emerging diseases.

# **MATERIALS AND METHODS PLANT MATERIALS**

*Arabidopsis* plants were grown in growth chambers with a 12 h light/12 h dark cycle, light intensity at 200 μmol m−2 s −1, 60% humidity, and 22◦C. The *ndr1-1* mutant was previously described (Century et al., 1995, 1997). Citrus plants,"Valencia" (*Citrus sinensis* [L.] Osbeck), were grown on the greenhouse bench and kept at 24◦C under natural light conditions. Plants were irrigated as needed and fertilized every 3 weeks using a water-soluble fertilizer mix, 20N–10P–20K (Peters Professional, The Scotts Company, Marysville, OH, USA).

# **BIOINFORMATIC ANALYSIS**

Basic Local Alignment Search Tools (BLAST) was used to search protein sequence databases for *Arabidopsis*1 and *Citrus sinensis*2, using appropriate query sequences. Sequence alignment and phylogenetic analysis were performed with the MEGA program (version 5.05). To construct the phylogenetic tree, the neighbor-joining method with 1000 bootstrap replications was used.

# **PATHOGEN INFECTIONS**

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*Pseudomonas syringae* strains used in this study were previously described (Lee et al., 2008; Wang et al., 2011b). Bacteria were cultured at 28◦C with King's B medium (10 g proteose peptone, 1.5 g K2HPO4, 3.2 ml 1 M MgSO4, and 5 g glycerol/l) containing the appropriate antibiotics. Freshly cultured bacteria at the optical

1http://blast.ncbi.nlm.nih.gov/Blast.cgi

2http://www.phytozome.net/search.php?method=Org\_Csinensis

density of 0.5–0.8 were harvested, washed once, and resuspended in 10 mMMgSO4 to make the infection solution at the desired concentrations. The fifth to seventh leaves of 25-day-old *Arabidopsis* plants were infected by infiltration with a 1-ml needleless syringe. For bacterial growth assay, six leaves selected from over 10 plants of each genotype were collected 3 days post-infiltration, bored with a core borer (6 mm in diameter), and ground for bacterial growth measurement as described previously (Lu et al., 2003). For hypersensitive response (HR) test, one-half of a leaf at the fifth, sixth, or seventh position was infiltrated with *P. syringae* pv. *maculicola* ES4326 *avrRpt2* (*Pma avrRpt2*; OD600 = 0.1) and scored 16–24 h post-infiltration for leaf collapse. Leaves infiltrated with 10 mM MgSO4 or the isogenic virulent strain *Pma* (OD600 = 0.1) were used as controls. At least 16 leaves from different plants of each genotype were scored for the HR symptoms. The rate of HR was expressed by the percentile of the number of leaves that developed HR symptoms out of the total number of inoculated leaves.

*Hyaloperonospora arabidopsidis* isolate Noco2 was a kind gift from S. Xiao at University of Maryland College Park. Strain propagation and preparation were conducted as previously described (Song et al., 2004; McDowell et al., 2010). *Hpa Noco2* spores (5 × 104 spores/ml in water) were sprayed on 7-day-old soilgrown seedlings. Sporangiophores on both sides of cotyledons were counted 7 days post-inoculation. At least 50 cotyledons from each genotype were counted to derive the average number of sporangiophores per genotype.

For *Ca.* L. asiaticus infection, 15-month-old "Valencia" plants were inoculated by grafting with two bark- or bud-pieces and two leaf pieces from infected greenhouse-grown "Valencia" plants, which were tested PCR-positive for *Ca.* L. asiaticus and demonstrated symptoms for HLB. Plants similarly inoculated but with disease-free tissue pieces obtainedfrom healthy greenhouse-grown "Valencia" plants were used as controls. Plants were pruned immediately after graft-inoculation to promote new leaf growth and HLB disease development. The inoculated plants were randomized periodically on the greenhouse bench to minimize the effect of environment on their defense responses to *Ca.* L. asiaticus.

#### **PCR-DETECTION OF** *Ca***. L. asiaticus IN CITRUS**

*Ca.* L. asiaticus-infected "Valencia" plants began to show typical HLB symptoms, yellowing and blotchy mottling around 11 weeks after inoculation (wai), which progressed more severely later. The symptomatic leaves were collected at 11 and 16 wai and extracted for DNA followed by PCR-detection of *Ca.* L. asiaticus (Albrecht and Bowman, 2012). Specifically, 100 mg leaf tissue was ground for DNA extraction, using the Plant DNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. For detection of *Ca.* L. asiaticus, real-time PCR assays were performed using primers HLBas and HLBr and the probe HLBp as described (Li et al., 2006). Amplifications were performed over 40 cycles using an ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA) and the QuantiTect Probe PCR Kit (Qiagen) according to the manufacturer's instructions. All reactions were carried out in a 20-μl reaction volume using 5 μl DNA. Once a branch was confirmed positive for *Ca.* L. asiaticus, symptomatic leaves on the branch were harvested for further RNA and SA analyses.

# **RNA EXTRACTION AND ANALYSIS**

Twenty-five-day-old *Arabidopsis* plants were harvested for RNA extraction and northern blotting as described (Ng et al., 2011). Radioactive probes were made by PCR with an antisense primer specific for a gene fragment in the presence of [32P] dCTP. Primers used for making the *CsNDR1* probe were CsNDR1- F1 (ATGTCAGAAAACGCCGGTG) and CsNDR1-R1 (TTAAG-CAAAAATCAAGACAAAAAAATAC). Primers for the *PR1* and *18S rRNA* probes were described previously (Ng et al., 2011).

Fully expanded leaves from *Ca.* L. asiaticus-infected "Valencia" plants showing HLB symptoms were collected 16 wai. One gram leaf tissue was ground in liquid nitrogen with a mortar and pestle and resuspended in 10 ml guanidinium isothiocyanate buffer (Chomczynski and Sacci, 1987). Total RNA was extracted as previously described (Strommer et al., 1993) with slight modifications. Phenol/chloroform/isoamyl alcohol (25:24:1) extraction was followed by two extractions with chloroform/isoamyl alcohol and precipitation of RNA with isopropanol at −20◦C overnight. RNA was pelleted by centrifugation at 10,000 *g* and 4◦C for 1 h, resuspended in 5 ml water and precipitated overnight at 0◦C with an equal volume of 8 M LiCl. After centrifugation at 10,000 *g* and 4◦C for 1 h, RNA was washed twice with 70% ethanol, air-dried, and dissolved in 500 μl of water. RNA was further purified, using the RNeasy- MinElute Cleanup kit (Qiagen) according to the manufacturer's instructions. Total RNA was DNase-treated using the TURBO DNA-*free*-KitTM (Ambion, Austin, TX, USA) according to the manufacturer's instructions.

Quantitative reverse transcription real-time PCR (qRT-PCR) was performed using an ABI 7500 real-time PCR system (Applied Biosystems) and the QuantiTect SYBR Green RT-PCR Kit (Qiagen) according to the manufacturer's instructions. Sixty nanograms of DNase-treated RNA were used in a total volume of 20 μl. For detection of *CsNDR1* transcripts, forward primer 5 -TGCTGCAGCTTCATCTTCAC-3 and reverse primer 5 -TGTCGTGTTGTTTCGGTTGT-3 were used. For detection of *18S rRNA* transcripts, forward primer 5 -GCTTAGGCCAAGGAAGTTTG-3 and reverse primer 5 - TCTATCCCCATCACGATGAA-3 were used. Melting curve analysis was performed to ensure amplification of a single product and the absence of primer-dimers. For relative quantification of gene expression, the 2−--CT method was applied as previously described (Livak and Schmittgen, 2001), using cycle threshold (Ct) values of *18S rRNA* for normalization.

#### **cDNA AMPLIFICATION, DNA CONSTRUCTION, AND PLANT TRANSFORMATION**

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To obtain the full-length *CsNDR1* cDNA sequence, we used the SMARTerTM RACE cDNA Amplification Kit (Clontech) to make a cDNA library from RNA extracted from *Ca.* L. asiaticusinfected 15-month-old "Valencia" plants. Nested primers, NDR1- 5R-P1 (CACTTTCTGATCGGTCAGCGCAG) and NDR1-5R-P2 (CAATCACGGACGTGCCGATG), were used to amplify the 5 end missing sequence while NDR1-3R-P1 (CATCG-GCACGTCCGTGATTG) and NDR1-3R-P2 (CTGCGCTGACC-GATCAGAAAGTG) were used to amplify the 3 end missing sequence. The amplified fragments were cloned in the pJET cloning vector, using the CloneJETTM PCR Cloning Kit (Thermo Scientific), and sequenced to obtain the full-length cDNA sequence. The full-length *CsNDR1* cDNA was further amplified from the library with NDR1-F1 (ATGTCAGAAAACGCCGGTG) and NDR1-R1 (TTAAGCAAAAATCAAGACAAAAAAATAC) and cloned into the *pJET* vector. At least 10 individual colonies were prepared for DNA and analyzed by sequencing. The sequence with fewer polymorphisms, compared with the reference sequence, and a correct open reading frame was used as the template for further cloning into the binary vector *pBINplusARS* under the control of the *CAMV 35s* promoter. The construct was confirmed by sequencing and transferred to*Agrobacterium tumefaciens* for *ndr1- 1* transformation, using the floral dipping method (Clough and Bent, 1998). T0 seeds were selected for T1 plants on MS plates containing kanamycin, resistance to which was conferred by the binary vector. T1 transgenic plants were collected for seeds, which were further selected for homozygous T2 plants.

#### **ION LEAKAGE MEASUREMENT**

Leaves of 25-day-old *Arabidopsis* plants were infiltrated with a bacterial suspension *Pma avrRpt2* (OD600 = 0.1) with a bluntend syringe, using 10 mM MgSO4 treatment as a control. At 0, 4, and 7 h post-inoculation, five leaf disks, cut with a 6-mm core borer, were collected, washed in de-ionized water, and placed in a 15-ml tube with 5 ml of de-ionized water. Triplicate tubes for each sample were gently shaken for 15 min followed by the measurement of solution conductivity, using an electrical conductivity meter (The London Company, Welwyn International Inc. Cleveland, OH, USA). Each tube was measured three times to derive average conductivity.

#### **SA MEASUREMENT**

Free and total SA (glucosylated SA) were extracted from 25 day-old Arabidopsis plants (Ng et al., 2011; Wang et al., 2011a). The same protocol was used to extract SA from leaves of *Ca.* L. asiaticus-positive "Valencia" plants that demonstrated HLB symptoms. SA separation and detection were conducted with a high-performance liquid chromatography (HPLC) instrument as previously described (Ng et al., 2011; Wang et al., 2011a).

#### **RESULTS**

#### **IDENTIFICATION OF A CITRUS NDR1 ORTHOLOG**

Since NDR1 plays a critical role in *Arabidopsis* defense, we set out to identify the citrus ortholog and investigate its role in defense regulation. In *Arabidopsis*, NDR1 belongs to a large protein family with over 40 members, named NDR1/HIN1-like (NHL) proteins (Dörmanna et al., 2000). BLAST searching of the sequence database of *Citrus sinensis*3 with the NDR1 protein sequence revealed a citrus protein (CsNDR1; orange1.1g028712m) with the highest similarity to NDR1 (the *E*-value is 2.4e−53) and three other top hits with *E*-values below 1.0 e−5. We further used CsNDR1 as the query to search the *Arabidopsis* protein database and retrieved sequences of NDR1 and 14 NHL proteins as the top hits. To determine the extent of similarity among these proteins, phylogenetic analysis was conducted, using the MEGA program (version 5.0; Tamura et al., 2011). **Figure 1** shows that CsNDR1 is in the same cluster with NDR1 with 99% bootstrap support. Two other citrus NHL proteins (orange1.1g041808m and orange1.1g08713m) are also in the same cluster with NDR1 but with lower confidence levels in bootstrap support. Thus, bioinformatic analysis suggests that CsNDR1 is an ortholog of NDR1.

#### **ECTOPIC EXPRESSION OF** *CsNDR1* **COMPLEMENTS** *Arabidopsis ndr1-1* **MUTANT**

To test if *CsNDR1* shares conserved function with its *Arabidopsis* correspondence, we used a genetic complementation approach. The full-length *CsNDR1* cDNA was amplified via RT-PCR from a cDNA library made from *Ca.* L. asiaticus-infected "Valencia" plants and was cloned initially to the pJET vector and then to the binary vector *pBINplusARS* under the control of the CAMV 35s promoter. The *CsNDR1*/*pBINplusARS* construct was used to transform *ndr1-1* via the standard floral dipping method (Clough and Bent, 1998). The presence of the transgene was confirmed by PCR with gene-specific primers. Initial infection of the T1 transgenic plants with a virulent strain *P. syringae* pv. *maculicola* ES4326 (*Pma*) indicated that some of the transgenic plants were more resistant than *ndr1-1* (data not shown)*.* We further isolated homozygous plants for eight independently transformed lines (*ndr1-1* + *CsNDR1)*. Infection of these plants with *Pma* showed that all lines were more resistant than *ndr1-1* and some were even more resistant than Col-0 (**Figure 2A**). Total RNA was isolated from these plants and northern blotting indicated that the level of disease resistance in some transgenic plants was correlated with the degree of transgene expression (**Figure 2B**). Thus, our results suggest that *CsNDR1* positively regulates *Arabidopsis* defense.

The *Arabidopsis* RPS2 is a CC–NBS–LRR type R protein. When recognizing the avirulent strain *Pma avrRpt2*, RPS2 activates strong defense responses. Such defense activation requires the function of NDR1 and sometimes leads to HR, a rapid programed cell death in the infected region (Aarts et al., 1998). We found that all transgenic plants showed enhanced disease resistance to *Pma avrRpt2* (OD600 = 0.0004), compared with *ndr1-1* (**Figure 3A**). In addition, the *ndr1-1* mutant showed compromised HR in response to a high dose of *Pma avrRpt2* (OD600 = 0.1), as indicated by the lack of leaf collapse (**Figure 3B**, top panel; Century et al., 1995). We found that all transgenic plants showed partial to full rescue of HR-defect of *ndr1-1* (**Figure 3B**, bottom panel).

Interestingly, line 15 that showed the highest level of *CsNDR1* expression, had a low frequency of leaf collapse in the HR assay, albeit still more than the *ndr1-1* mutant. We also noticed that this line is smaller than other lines (**Figure 4A**). Thus we suspected that the small leaf size might obscure the HR scoring. To better quantify the HR cell death, we performed ion leakage measurement with this line and another line (#9) that showed medium *CsNDR1* expression (**Figure 2B**). When challenged with *Pma avrRpt2* (OD600 = 0.1), *ndr1-1* had the lowest level of ion leakage (**Figure 4B**; Zhang et al., 2004), consistent with its HR-deficit. The ion leakage level was highest in line 15 and medium in line 9, compared with Col-0. Together disease resistance and HR assays suggest that *CsNDR1* functions similarly as

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3http://www.phytozome.net

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**FIGURE 1 | Neighbor-joining phylogenetic tree to show relationship among NDR1 homologs from** *Arabidopsis* **and citrus.** The protein sequences were retrieved from BLAST search of *Citrus sinensis* protein database with NDR1 sequence as a query or from BLAST search of *Arabidopsis* protein database with CsNDR1 sequence as a query, using an *E*-value cutoff e−4. The MEGA program (version 5.05) was used to construct the tree. Protein sequences of the indicated genes were aligned with the ClustalW method and the tree was generated with the neighbor-joining method, using 1000 bootstrap replications. Numbers on the tree indicate bootstrap support (values <50% not shown). Branch lengths were drawn to scale; size bar represents number of amino acid substitutions per site. RIN4 protein sequence was used as an outgroup to root the tree.

*Arabidopsis NDR1* in both basal and resistance protein-mediated defense.

#### **ECTOPIC EXPRESSION OF** *CsNDR1* **LEADS TO ACTIVATION OF SA-MEDIATED DEFENSE AND BROAD-SPECTRUM DISEASE RESISTANCE**

Salicylic acid is a key signaling molecule regulating defense pathways including basal defense, R gene-mediated resistance, and systemic acquired resistance (Durrant and Dong, 2004; Lu, 2009). To test whether SA-mediated defense is activated in the transgenic plants, we quantified SA levels. We found that line 15 but not line 9 accumulated much higher levels of both free and total SA (glucosylated SA; **Figure 4C**). Consistent with its high SA levels, line 15 also showed higher expression of the SA marker gene *PR1* (**Figure 4D**). These results suggest that overexpression of *CsNDR1* to a certain level activates SA signaling.

To further investigate how overexpressing *CsNDR1* affects disease resistance, we challenged line 9 and 15 with additional *P. syringae* strains. Ectopic expression of *CsNDR1* complemented susceptibility conferred by *ndr1-1* to the virulent strain *P. syringae* pv. *tomato* DC3000 (*Pto*; **Figure 5A**, left). Line 15 is also more resistant to the isogenic avirulent strain *Pto avrRps4* (**Figure 5A**, right), which is recognized by RPS4 (a TIR–NBS–LRR type of R protein) independently of NDR1 (Aarts et al., 1998). Thus, these results indicate that ectopic expression of *CsNDR1* leads to activation of resistance to a pathogen that the endogenous gene otherwise does not have an effect on. In addition, we found that line 9 and 15 showed increased resistance to the virulent oomycete

samples (*P* < 0.05). **(B)** Expression analysis of transgenic plants carrying *CsNDR1*. Total RNA was extracted from 25-day-old plants and analyzed by northern blotting. A probe specific to *CsNDR1* was used to detect expression of the transgene. The *18S rRNA* probe was used to indicate equal loading in the samples. These experiments were repeated two times with similar results.

pathogen *Hpa* Noco2, compared with *ndr1-1* (**Figure 5B**). Thus, overexpressing *CsNDR1* could lead to broad-spectrum disease resistance.

# **SA ACCUMULATION AND EXPRESSION OF** *CsNDR1* **ARE MODESTLY INDUCED BY** *Ca***. L. asiaticus INFECTION**

To see how SA signaling is affected by HLB in citrus, we infected 15-month-old "Valencia" plants with *Ca.* L. asiaticus, using mocktreated plants as a control. We began to observe at 11 wai HLB symptoms, chlorosis and/or blotchy mottling of leaves, which increased in severity by 16 wai (**Figure 6A**). We did qPCR with the symptomatic and control leaves, using primers specific to *Ca.* L. asiaticus. The average Ct values of the symptomatic leaves were 21.8 at 11 wai and 20.7 at 16 wai. No *Ca.* L. asiaticus was detected in the control. Thus these symptomatic leaves were confirmed to be *Ca.* L. asiaticus positive. The symptomatic leaves were further collected for SA measurement and RNA analysis. Compared with the mock control, the symptomatic leaves from infected plants had about twofold more total SA levels (**Figure 6B**). Similarly, expression of *CsNDR1* was also induced about twofold more in the infected leaves (**Figure 6C**). These data suggest that SA and/or

(OD600 = 0.0004). Bacterial growth was assessed 3 days after infection. Data represent the average of bacterial numbers in six samples ± standard error. Statistical analysis was performed with log transformed data, using one-way ANOVA Fisher's PLSD tests (StatView 5.0.1). Different letters indicate significant difference among the samples (*P* < 0.05). **(B)** HR test. Half leaves of the fourth to seventh leaves of 25-day-old plants were infiltrated with *Pma avrRpt2* (OD600 = 0.1) and scored for the HR symptoms 16 h post-infection. At least 16 leaves were infected in each sample. *Top panel*: picture of infected leaves. The arrowheads indicate leaf collapse, a typical HR symptom, in Col-0 and a representative complemented plant (line 9) but not in *ndr1-1*. *Bottom panel*: the percentage of the infected leaves of each genotype that showed HR. These experiments were repeated two times with similar results.

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Other transgenic lines resemble Col-0 (not shown). **(B)** Ion leakage measurement. The fourth to seventh leaves of 25-day-old plants were infiltrated with *Pma avrRpt2* (OD600 = 0.1) and collected at the indicated times post-infection for ion leakage measurement. The data represent average of triplicate samples, each of which contained five individual leaf disks of 6 mm in diameter. **(C)** SA quantitation. Twenty-five-day-old plants were harvested for SA extraction followed by HPLC analysis. Asterisks indicate significant difference between the samples and Col-0 (*P* < 0.05; *n* = 3). **(D)** Northern blotting. DNA fragments specific to *PR1* and *18s rRNA*, respectively, were used to probe blots containing RNA samples from Col-0, *ndr1-1*, and transgenic lines 9 and 15. These experiments were repeated two times with similar results.

*CsNDR1*-mediated signaling are activated modestly upon *Ca.* L. asiaticus infection.

#### **DISCUSSION**

In this study, we presented bioinformatic and experimental evidence suggesting that the citrus gene *CsNDR1* is an *Arabidopsis NDR1* ortholog. Overexpression of *CsNDR1* in *Arabidopsis*

rescues *ndr1-1*-conferred susceptibility to *P. syringae* infection and leads to broad-spectrum disease resistance to the oomycete pathogen *Hpa* Noco2. We also found that both SA accumulation and *CsNDR1* expression are induced to modest levels in citrus upon infection with *Ca.* L. asiaticus, the agent associated with HLB. Our data suggest a possibility that manipulation of SA/*CsNDR1*-mediated defense may lead to enhanced resistance to HLB and other devastating diseases in citrus.

SA plays a critical role in regulating plant resistance against various pathogens. Broad-spectrum disease resistance has been successfully introduced into several economically important plants via manipulation of the SA pathway. Much of the previous studies have been focused on NPR1, a key SA signal transducer. For instance, overexpression of *Arabidopsis* NPR1 and/or its homologs from other plants confers resistance against diverse bacterial and fungal pathogens in *Arabidopsis*, apple, citrus, soybean, tomato,

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results.

rice, and/or wheat (Fitzgerald et al., 2004; Li et al., 2004; Makandar et al., 2006; Malnoy et al., 2007; Sandhu et al., 2009; Zhang et al., 2010). These observations suggest that SA-mediated defense is conserved in monocots and dicots, and can be activated to against a range of pathogens (Campbell et al., 2002). Consistent with this notion, reports showed that exogenous application of SA agonists, such as benzothiadiazole and its commercial forms, acibenzolar-*S*-methyl (ASM, Actigard-, Syngenta Crop Protection, Inc.) and imidacloprid (Imid, Admire-, Bayer Crop Science), to citrus and other plants could induce defense marker gene expression and/or activate some protections of plants to a variety of viral, bacterial, and fungal pathogens (Campbell et al., 2002; Maxson-Stein et al., 2002; Dekkers et al., 2004; Francis et al., 2009; Graham and Myers, 2009).

*Arabidopsis NDR1* is a positive SA regulator, which is known to be specifically required for defense activated by some CC– NBS–LRR R proteins but not by some TIR–NBS–LRR type of R proteins (Aarts et al., 1998; Coppinger et al., 2004). Although not much is known about R-avr recognition in many non-*Arabidopsis* plants, the fact that *NDR1* homologs widely exist in diverse plants

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(Lee et al., 2006; Chong et al., 2008; Cacas et al., 2011) suggests conserved defense signaling involving *NDR1* homologs. Recently a coffee *NDR1* homolog was shown to complement the *ndr1-1* mutant for its susceptibility to *P. syringae* strains (Cacas et al., 2011). Here we also showed a complementation of *ndr1-1* by *CsNDR1*. The transgenic plants showed varying levels of *CsNDR1* expression, which is not uncommon for transgenes (Lu et al., 2003). We noticed that there is a degree of correlation between the level of transgene expression and the level of disease resistance in some transgenic plants (**Figure 2**). One line highly expressing *CsNDR1* (line 15) constitutively activates SA-mediated defense, associated with enhanced disease resistance to *Pto avrRps4* (that is not affected by the endogenous *Arabidopsis* NDR1) and to the oomycete isolate *Hpa Noco2* (**Figure 5**). It is known that signals induced by different R genes upon recognition of their cognate effectors from pathogens can converge at downstream steps, involving SA-mediated defense (Martin et al., 2003; Chisholm et al., 2006). Our data suggest that hyper-activation of one branch of R-gene pathways, such as the *CsNDR1* branch, could potentially activate SA signaling, leading to broad-spectrum disease resistance.

In *Arabidopsis*, both resistant and susceptible responses to pathogen infection are characterized by elevated SA accumulation and defense gene induction but with differences in the speed and amplitude of the responses (Zhou et al., 1998; Maleck et al., 2000; Tao et al., 2003; Song et al., 2004). Compared with a resistance response in *Arabidopsis*, induction of SA levels in citrus infected with *Ca.* L. asiaticus is quite small (**Figure 6B**). In addition, our gene expression data (**Figure 6C**) and microarray analyses (Albrecht and Bowman, 2008, 2012) indicate that the spectrum and intensity of defense genes induced by *Ca.* L. asiaticus are also quite limited. These observations suggest that when infected by *Ca.* L. asiaticus, citrus plants do not activate considerable host defense. This can be explained with at least two possible reasons: (1) a lack of recognition of effector proteins from *Ca.* L. asiaticus by citrus; and/or (2) a suppression of host defense by *Ca.* L. asiaticus. Thus, the interaction between citrus and *Ca.* L. asiaticus can be viewed as a compatible interaction, leading to disease symptom development in the host. The relatively low level of host defense in response to *Ca.* L. asiaticus also suggests a possibility that HLB resistance can be achieved if we could manipulate the host to enhance its defense levels.

Genetic engineering is a particularly attractive approach to introduce disease resistance traits into citrus because citrus has long juvenile growth – it typically takes 5–15 years for a citrus plant to flower. In addition, most commercial citrus cultivars produce polyembryonic seeds asexually, which complicates the process of introducing novel traits into citrus via traditional breeding (Koltunow et al., 1996). Recently the *Arabidopsis NPR1* gene was shown to increase resistance to the canker disease when overexpressed in citrus (Zhang et al., 2010). Thus, *CsNDR1*, citrus *NPR1*, and other citrus homologs of SA regulatory genes are ideal candidates that can be genetically manipulated to increase their expression in order to test if these genes confer resistance to HLB in citrus. Engineering such genes could yield citrus plants with enhanced disease resistance that are also more acceptable to the consumers than those engineered with similar genes from other plants. Moreover, the newly released citrus genome sequence has greatly facilitated the identification of additional citrus defense genes. We anticipate that large-scale functional genomic analysis could uncover defense genes that play critical roles in resistance against HLB, citrus canker, and/or other emerging diseases challenging the citrus industry worldwide.

# **REFERENCES**

# **ACKNOWLEDGMENTS**

We thank members in the Lu laboratory for assistance with this work. We thank Dr. Kevin Omland for assistance in phylogenetic analysis, Dr. William LaCourse for sharing his HPLC instrument, and Mr. Tim Ford for taking pictures for this publication. This work was supported by a grant from Citrus Research and Development Foundation to Hua Lu and a scholarship from China Scholarship Council to Chong Zhang.

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hemisphere. *Outlooks Pest Manage.* 18, 274–279.

Transgenic tomato plants expressing the *Arabidopsis NPR1* gene display enhanced resistance to a spectrum of fungal and bacterial diseases. *Transgenic Res.* 13, 567–581.

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 20 February 2013; accepted: 07 May 2013; published online: 03 June 2013.*

*Citation: Lu H, Zhang C, Albrecht U, Shimizu R, Wang G and Bowman KD (2013) Overexpression of a citrus NDR1 ortholog increases disease resistance in Arabidopsis. Front. Plant Sci. 4:157. doi: 10.3389/fpls.2013.00157*

*This article was submitted to Frontiers in Plant-Microbe Interaction, a specialty of Frontiers in Plant Science.*

*Copyright © 2013 Lu, Zhang, Albrecht, Shimizu, Wang and Bowman. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any thirdparty graphics etc.*

**MINI REVIEW ARTICLE** published: 18 March 2014 doi: 10.3389/fpls.2014.00101

# Cell wall methanol as a signal in plant immunity

# *Tatiana V. Komarova1,2 , Ekaterina V. Sheshukova2 andYuri L. Dorokhov1,2 \**

*1 A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia 2 N. I. Vavilov Institute of General Genetics, Russian Academy of Science, Moscow, Russia*

#### *Edited by:*

*Vincenzo Lionetti, Sapienza Università di Roma, Italy*

#### *Reviewed by:*

*Simone Ferrari, Sapienza Università di Roma, Italy Alessandro Raiola, University of Padova, Italy*

*\*Correspondence:*

*Yuri L. Dorokhov, A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119991, Russia e-mail<EMAIL_ADDRESS>
Cell wall pectin forms a matrix around the cellulose–xyloglucan network that is composed of rhamnogalacturonan I, rhamnogalacturonan II, and homogalacturonan (HG), a major pectic polymer consisting of α-1,4-linked galacturonic acids. HG is secreted in a highly methylesterified form and selectively de-methyl-esterified by pectin methylesterases (PMEs) during cell growth and pathogen attack. The mechanical damage that often precedes the penetration of the leaf by a pathogen promotes the activation of PME, which in turn leads to the emission of methanol (MeOH), an abundant volatile organic compound, which is quickly perceived by the intact leaves of the damaged plant, and the neighboring plants.The exposure to MeOH may result in a "priming" effect on intact leaves, setting the stage for the within-plant, and neighboring plant immunity. The emission of MeOH by a wounded plant enhances the resistance of the non-wounded, neighboring "receiver" plants to bacterial pathogens and promotes cell-to-cell communication that facilitates the spread of viruses in neighboring plants.

**Keywords: cell wall, methanol, pectin, pectin methylesterase, plant immunity, priming**

# **INTRODUCTION**

Plant cells are covered with a dense extracellular matrix that prevents direct contact between adjacent cells and pathogens. Damage to the plant epidermis caused by abiotic (wind, hail, and rain) and biotic (insects) factors may allow the penetration of pathogens (bacteria, fungi, oomycetes, and nematodes) into the intercellular space of the leaf and virus particles into the cell. Thus, plant wounding is one of the conditions for pathogen entry. However, mechanical damage to the leaf promotes the emission of volatile organic compounds (VOCs), including the green leaf volatiles (GLVs), and methanol (MeOH), which are quickly perceived by the intact leaves of the damaged plant and the intact neighboring plants (Dorokhov et al., 2014). The transport of VOCs is much faster compared to the transport of infectious viral entities and bacterial effectors through the phloem. Thus, exposure to VOCs may result in a "priming" effect on intact leaves, setting the stage for subsequent plant immunity. GLVs are associated with the smell of a freshly mown lawn and are derived from C18 fatty acids released from damaged membranes. MeOH, quantitatively the most important plant volatile after CO2, is a product of the demethylation of pectin by the pectin methylesterases (PMEs) during cell wall (CW) formation and modification (Pelloux et al., 2007).

# **PME-MEDIATED PLANT IMMUNITY**

Cell wall pectin forms a matrix around the cellulose–xyloglucan network that is composed of three main components called rhamnogalacturonan I (RGI), rhamnogalacturonan II (RGA II), and homogalacturonan (HG), a major pectic polymer consisting of α-1,4-linked galacturonic acids (Peaucelle et al., 2012). HG is secreted in a highly methyl-esterified form and selectively demethyl-esterified by PMEs, resulting in MeOH formation. The *PME* genes encode a pro-PME precursor with an *N*-terminal

extension of variable length that is essential for protein targeting to the endoplasmic reticulum (Dorokhov et al., 1999). PME maturation requires removal of the PME leader including the transmembrane domain and spacer sequence (Dorokhov et al., 2006a). It was hypothesized that the spacer sequence plays a role in subcellular targeting and acts as an intramolecular chaperone for folding of the mature enzyme or as an autoinhibitor during transport through the endomembrane system (Pelloux et al., 2007). PME participates in CW modulation during general plant growth as it is involved in cell expansion and CW modification (Pelletier et al., 2010). The synthesis of PME is one of the aspects of plant growth that leads to the demethylesterification of the elastic "soft" pectins that accompanies MeOH generation (Komarova et al., 2014) as part of the natural division and maturation of the plant cell. After demethylesterification, pectate can form Ca2+ pectate cross-linked complexes of rigid "hard" pectin, referred to as "egg boxes" (Peaucelle et al., 2012).

The important role of PME in the resistance of plants to fungi and bacteria has been demonstrated (Pelloux et al., 2007). A higher degree of pectin methyl esterification in certain plants induces resistance to pathogenic fungi (Lionetti et al., 2012). CW pectin methyl esterification may have an impact on plant resistance because highly methyl-esterified pectin can be less susceptible to hydrolysis by pectic enzymes such as fungal endopolygalacturonases. This view is supported by experiments performed with plants that were stably transformed with the *PME inhibitor (PMEI)* gene. The PMEI transgenic *Arabidopsis* (Lionetti et al., 2007) and durum wheat (Volpi et al., 2011) plants exhibited high levels of resistance to fungal and bacterial pathogens. Moreover, PME-mediated pectin methyl de-esterification may influence the polygalacturonase-mediated release of pectin-derived compounds, which in turn elicits a defense response (Pelloux et al., 2007; Lionetti et al., 2012).

The role of PME in viral infection is more complicated. PME interacts with the movement protein (MP) of the *Tobacco mosaic virus* (TMV; Dorokhov et al., 1999; Chen et al., 2000), suggesting that PME may be involved in the cell-to-cell movement of plant viruses (Chen and Citovsky, 2003). Interestingly, PMEI also interacts with PME to negatively affect viral infection (Lionetti et al., 2014), most likely by interfering with PME and TMV MP binding. The complex role of PME in viral infection is also underscored by the effects of PME on nuclear protein transport (Komarova et al., 2011) and gene silencing mediated by the activation of siRNA and miRNA production (Dorokhov et al., 2006b).

#### **MeOH AND PLANT IMMUNITY**

The PME-mediated conversion of HG methoxyl groups into carboxyl groups results in MeOH release. In humans, MeOH is considered to be a poison because alcohol dehydrogenase metabolizes MeOH into toxic formaldehyde. However, recent data have indicated that MeOH is actually a naturally occurring compound in normal, healthy human individuals. MeOH is not toxic to plant cells and has long been assumed to be a metabolic waste product. Recently, it has been shown that MeOH may regulate plant growth (Komarova et al., 2014) and serve an alarm function (Dorokhov et al., 2012a). The effects of PME-generated MeOH emitted from plants ("emitters") on the defensive reactions of other plants ("receivers") were studied (Dorokhov et al., 2012a). The results of this study led to the conclusion that MeOH is a signaling molecule that is involved in within-plant and plant-to-plant communication (Dorokhov et al., 2012a).

Mechanical damage to plants drastically increases MeOH and GLVs emission. GLVs that are rapidly released from wounded leaves may in turn stimulate PME-generated MeOH production (Dorokhov et al., 2012a). Herbivore attacks also increase MeOH emission levels: *Manduca sexta* caterpillars enhance woundinduced MeOH emission in *Nicotiana attenuate* (von Dahl et al., 2006). The over-expression of PME, derived from *Arabidopsis thaliana* and *Aspergillus niger*, in transgenic tobacco plants enhances resistance to polyphagous insect pests (Dixit et al., 2013). Transgenic plants with a silenced *PME* gene exhibited a 50% reduction in PME activity in their leaves and a 70% reduction in herbivore-induced MeOH emissions compared to wild type plants. This result demonstrates that herbivore-induced MeOH emissions originate from pectin demethylation by PME (Körner et al., 2009). The emission of MeOH is very fast and can be detected immediately following mechanical damage. Thus, the MeOH emitted from wounded leaves is produced by two forms of PME: pre-existing PME deposited in the CW before wounding, which allows rapid MeOH release (Körner et al., 2009), and PME that is synthesized *de novo* after wounding (Dorokhov et al., 2012a), which likely generates MeOH for an extended period.

Unlike longer-chain alcohols, the MeOH emitted by a wounded plant attracts insects and bark beetles. Moreover, mice prefer the odor of MeOH to the odors of other plant volatiles under laboratory conditions, and MeOH exposure alters the accumulation of mRNA in the mouse brain (Dorokhov et al., 2012b). This finding led to the conclusion that the MeOH emitted by wounded plants may have a role in plant-animal signaling.

Investigations demonstrated (Dorokhov et al., 2012a) that increased MeOH emissions from *PME*-transgenic or mechanically wounded non-transgenic plants retarded the growth of the bacterial pathogen *Ralstonia solanacearum* in neighboring "receiver" plants. The suppression of *R. solanacearum* growth observed in the "receiver" plants could be caused by gaseous MeOH or/and by GLVs. Indeed, *cis*-3-hexen-1-ol evaporated in a desiccator also resulted in decreased bacterial growth in the target plants. However, GLVs rapidly released from wounded leaves stimulated PME-generated MeOH production (Dorokhov et al., 2012a), suggesting that their influence on bacterial growth may be indirect. MeOH-stimulated antibacterial resistance was preceded by the upregulation of genes that control stress response and cell-to-cell communication in the "receiver". Antibacterial resistance accompanied by MeOH-induced genes (MIGs) upregulation was most likely related to the transcriptional induction of the *type II proteinase inhibitor (PI-II)* gene. PI-IIs are powerful inhibitors of serine endopeptidases in animals and microorganisms (Turra and Lorito, 2011). The *PI-II* gene is not expressed in the leaves of healthy plants, but it is induced in leaves that have been subjected to different types of stress, including wounding and bacterial infection. *PME*-transgenic tobacco with high levels of *PI-II* expression exhibited increased resistance to *R. solanacearum* (Dorokhov et al., 2012a). This finding supports the role of *PI-II* in the suppression of bacterial proteases.

Experiments with gaseous MeOH provided examples of priming in intact plants (**Figure 1**), which led to conditions conducive for viral infection (Dorokhov et al., 2012a). This effect could be explained by the enhancement of cell-to-cell communication by the MIGs, such as β*-1,3-glucanase* (*BG*; Zavaliev et al., 2011) and *non-cell-autonomous pathway protein* (*NCAPP*; Lee et al., 2003).

A model (**Figure 1**) proposing that MeOH-triggered PD dilation should enhance viral spread within the plant was confirmed in experiments in which BG and NCAPP activated cell-to-cell communication and TMV RNA accumulation. Moreover, gaseous MeOH or the vapors from wounded plants increased TMV reproduction in the "receivers" (Dorokhov et al., 2012a).

Thus, MeOH has a contradictory effect on the sensitivity of the leaves of the "receiver" plant to bacteria and viruses. The mechanisms that underlie this phenomenon are not clear; however, we can consider two factors that may explain this inconsistency in the MeOH-induced effects. First, there is a fundamental difference between bacteria and viruses with respect to their modes of intercellular transport. Bacterial pathogens do not cross the plant CW boundaries because they inhabit the intercellular spaces. In contrast, viral pathogens require cell-to-cell movement for local and systemic spread. Second, the most abundant MIGs can be divided into two groups according to their ability to participate in either bacterial or viral pathogenesis. The first, including *PI-II* and *PME* inhibitor, are involved in immunity against non-viral pathogens. The second group of genes, including *NCAPP* and *MIG-21* (Dorokhov et al., 2012a), is involved in the PD-mediated intercellular transport and reproduction of viruses. The most

abundant MIG, the *BG* gene, is involved in antibacterial immunity; however, the BG protein also accelerates PD-mediated intercellular transport.

# **CONCLUSION**

Based on the available data, we can conclude that woundingstimulated MeOH that is released into the air by damaged plants or plants compromised by herbivorous insects serves as an alarm to help neighboring plants or adjacent leaves prepare for a defense. The MeOH provides protection against herbivorous insects and plant pathogens such as bacteria. However, considering the role of MeOH in the relationship between viruses and plants, we do not find a negative, or even a neutral, influence of MeOH on viruses. On the contrary, the findings described (Dorokhov et al., 2012a) indicate that MeOH sensitizes the plant to allow the entry and spread of a virus through the plant and between plants by insect vectors. Therefore, MeOH promotes viral propagation. The positive impact of MeOH on viral infection may be explained by several factors. First, plant viruses differ from other types of pathogens as they inhabit the symplast. Furthermore, the survival of a virus depends on its ability to move from cell-to-cell exploiting PD to accumulate to sufficient levels and in enough tissues to guarantee survival despite using a very limited amount of genetic material. Thus, a virus, with its small but highly variable genome, spends its entire life in the cell symplast, while other pathogens occupy the apoplast. Second, the symplast is not only the space in which viruses reproduce, but it is also the site of RNA interference mechanisms that serve to eliminate foreign RNA. The specific degradation of RNA by RNA interference allows the host plant to effectively control viruses and other pathogens. It is known that the intracellular and intercellular transport of

silencing factors is necessary for effective RNA interference. Therefore, a MeOH-mediated increase in viral replication may be regarded as compensation for the acquisition of antimicrobial resistance.

# **ACKNOWLEDGMENTS**

This work was supported by the Russian Science Foundation, the Russian Foundation for Basic Research (grants Nos. 11-04-01152, 12-04-33016, 14-04-00109), and stipend of the President of the Russian Federation for young scientists.

#### **REFERENCES**

pectin methylesterase: the host-cell receptor for the tobacco mosaic virus movement protein. *FEBS Lett.* 461, 223–228. doi: 10.1016/S0014-5793(99) 01447-7

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 31 January 2014; accepted: 02 March 2014; published online: 18 March 2014. Citation: Komarova TV, Sheshukova EV and Dorokhov YL (2014) Cell wall methanol as a signal in plant immunity. Front. Plant Sci. 5:101. doi: 10.3389/fpls.2014.00101 This article was submitted to Plant-Microbe Interaction, a section of the journal Frontiers in Plant Science.*

*Copyright © 2014 Komarova, Sheshukova and Dorokhov. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited andthatthe original publication inthis journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Sucrose and invertases, a part of the plant defense response to the biotic stresses

# *Alexandra S. Tauzin† ‡ and Thierry Giardina\*‡*

*CNRS, Centrale Marseille, iSm2 UMR 7313, Aix Marseille Université, Marseille, France*

#### *Edited by:*

*Vincenzo Lionetti, Sapienza "Università di Roma," Italy*

#### *Reviewed by:*

*Zuhua He, Chinese Academy of Sciences, China Ingo Bergmann, Leibniz Institute for Agricultural Engineering Potsdam-Bornim e.V. (ATB), Germany*

#### *\*Correspondence:*

*Thierry Giardina, CNRS, Centrale Marseille, iSm2 UMR 7313, Aix Marseille Université, Avenue Escadrille Normandie-Niemen, 13397 Marseille, France e-mail<EMAIL_ADDRESS>
#### *†Present address:*

*Michael Smith, Laboratories and Department of Chemistry, University of British Columbia, Vancouver, Canada*

*‡These authors have contributed equally to this work.*

#### **INTRODUCTION**

Cash and subsistence crops are susceptible to a large number of diseases caused by plant pathogens. Among pathogenic organisms: fungi, oomycetes, viruses and bacteria are the most important ones. The direct consequence of pathogen attack is the decrease of the crop yield. In addition to economic loss, consumer health may be compromised due to risks in ingesting toxins produced from secondary metabolites of these pathogens. Mycotoxins are probably the most known factors produced by fungi, which are not only poisonous but also carcinogenic for human (Maresca, 2013).

The plant response is mediated by a sophisticated immune system divided into two different pathways. The first is microbialassociated molecular-patterns-triggered immunity (MTI), constituted by elicitors recognized by the plant innate immune systems *via* pattern recognition receptors (PRRs) (Ausubel, 2005; Katagiri and Tsuda, 2010). The second is the effector-triggered immunity (ETI) stimulated on the basis of the perception of pathogen effectors by plant disease resistance proteins (Dangl and Jones, 2001; Jones and Dangl, 2006).

Pathogens modify the host metabolism which results in an energy increase and production of carbon sources (Thines et al., 2000) including sucrose and its cleavage products, glucose and fructose (Roitsch and Gonzalez, 2004; Rolland et al., 2006). Sucrose hydrolysis is catalyzed by invertases, and the consequence is the shifts of the apoplastic sucrose/hexose

Sucrose is the main form of assimilated carbon which is produced during photosynthesis and then transported from source to sink tissues *via* the phloem. This disaccharide is known to have important roles as signaling molecule and it is involved in many metabolic processes in plants. Essential for plant growth and development, sucrose is engaged in plant defense by activating plant immune responses against pathogens. During infection, pathogens reallocate the plant sugars for their own needs forcing the plants to modify their sugar content and triggering their defense responses. Among enzymes that hydrolyze sucrose and alter carbohydrate partitioning, invertases have been reported to be affected during plant-pathogen interactions. Recent highlights on the role of invertases in the establishment of plant defense responses suggest a more complex regulation of sugar signaling in plant-pathogen interaction.

**Keywords: sucrose, cell wall invertase, vacuolar invertase, alkaline/neutral invertase, plant defense response**

ratio in favor of hexoses. The aim of this paper is to review recent evidence on the crucial roles of invertases during plant pathogen attacks and how the invertase activity is regulated.

# **FROM CARBOHYDRATE PARTITIONING TO PLANT DEFENSE RESPONSE**

#### **SUCROSE SIGNAL MOLECULE**

In higher plants, sucrose is the major transport form of carbohydrates. Sucrose is produced during photosynthesis in source tissues (leaves), and then transported *via* the phloem to the different sink tissues (roots, stem, reproductive organs and vegetative storage organs) to provide the carbon and energy needed for growth and synthesis of storage reserves.

The role of sucrose as signaling molecule is well established (for reviews see Koch, 2004; Rolland et al., 2006; Wind et al., 2010; Tognetti et al., 2013). It affects plant development processes such as plant growth, regulation of flowering, differentiation of vascular tissue and development of storage organs (for review see Tognetti et al., 2013). Sucrose cleavage products, glucose and fructose, also act as signaling molecules. Of the two hexoses, glucose has been better described in relation with the hexokinase signaling pathway (Moore et al., 2003; Cho et al., 2009) while for fructose a specific pathway has been proposed involving the abscisic acid (ABA)- and ethylene-signaling pathway (Cho and Yoo, 2011; Li et al., 2011).

Gomez-Ariza et al. (2007) observed that the pre-treatment of rice plants with sucrose drastically reduced symptoms of fungal *Magnaporthe oryzae* infection and they proposed sucrose as a signal molecule in plant immunity.

#### **PLANT INVERTASES**

Invertases (EC.<IP_ADDRESS>) hydrolyze irreversibly sucrose into glucose and fructose. Three groups were identified: alkaline/neutral invertases (A/NInv) localized in the cytosol, mitochondria and/or in plastids, and two types of acid invertases, insoluble bound to the cell wall (cell wall invertase, CWI) and soluble found in the vacuole space (vacuolar invertase, VI), respectively.

#### **ACID INVERTASES AND PROTEINACEOUS INHIBITORS**

Acid invertases, CWIs and VIs, belong to the GH32 family. CWIs play a key role in sucrose partitioning, plant development and cell differentiation while VIs are involved in cell expansion, sugar storage and regulation of cold induced sweetening (Roitsch and Gonzalez, 2004). Both are post-translationally regulated by proteinaceous inhibitors (INHs) which belong, with pectin methylesterase inhibitors (PMEIs), to the pectin methylesterase inhibitor related protein (PMEI-RP) family (Pfam 04043) (Hothorn et al., 2004).

During plant infection, the level of VI modulation is poorly understood with contradictory reports in the literature that leads to an unclear functional assignment (**Table 1**). On the one hand, a reduction of VI expression has been observed during the infection of *Vicia faba* by *Uromyces fabae* and *Vitis vinifera* by *Erysiphe necator* and *Plasmopora viticola* (Voegele et al., 2006; Hayes et al., 2010). This down-regulation was attributed to a decrease in the availability of sucrose in the storage compartment (Voegele et al., 2006; Hayes et al., 2010). By contrast, a high VI activity was observed during the first stage of infection of castor beans by *Agrobacterium tumefaciens* that might suggest a supportive function during invasion (Wachter et al., 2003). Moreover, the expression of a VI (TIV-1) is not affected in tomato infected by *Botrytis cinerea* (Hyun et al., 2011). Finally, when Essmann et al. compared wild type tobacco plants and transgenic plants silenced for CWI after infection by *Phytophthora nicotianae*, they noticed no significant changes in the VI activity (Essmann et al., 2008a,b) suggesting that the VI is not involved in the plant defense response. These results reinforce the doubts concerning the exact role of VIs in plant immunity.

By contrast, the link between plant response against pathogen and CWI activity has been widely studied (**Table 1**). A common trend is observed for the rapid increase of the CWI mRNA level after infection by bacterial, fungal, viruses, oomycetes and nematodes (for detailed references see **Table 1**). Indeed, the up-regulation of CWI activity is essential to modulate sugar partitioning and provide the sugars which are necessary for the pathogen development. A clear example has been demonstrated for gall development in *A. thaliana* (Siemens et al., 2011). Moreover, it was shown that during infection CWI activity also triggers plant defense responses such as induction of defenserelated gene expression, callose deposition and reduction of photosynthesis or cell death. CWI silencing disrupts the ability of transgenic plants to answer correctly to the pathogen attacks and impairs the defense induced reaction (Essmann et al., 2008a). In rice, the loss-of-function mutant of the CWI gene GRAIN INCOMPLETE FILLING 1 (GIF1) has been demonstrated to be hypersusceptible to postharvest pathogens while the constitutive expression of GIF1 enhances the resistance to pathogens by activating the plant defense response (Sun et al., 2013). In the particular case of symbiosis (such as arbuscular mycorrhiza), the expression of CWI is finely controlled by the partner to prevent the induction of *pathogenesis-related* (PR) genes and promote "long-term" interaction (Schaarschmidt et al., 2006, 2007).

Invertase activity is potentially modulated by proteinaceous inhibitors (INHs) in a pH-dependent manner (Tauzin et al., 2014). Greiner et al. (1998) demonstrated that tobacco INH didn't affect invertases purified from two fungi, *Candida utilis* and *Saccharomyces cerevisiae*, supporting the idea that INHs are not involved in plant defense mechanisms. However, a strong repression of the expression of one of the three INHs from *A. thaliana* after infection by *Pseudomonas syringae* pv. tomato DC3000 was documented (Bonfig et al., 2010). The invertase activity was detectable only in infected plants while the enzyme was present in infected and uninfected crude extract cells, indicating that the enzyme activity was repressed by a specific inhibitor. This result was corroborated by the utilization of the pseudo tetrasaccharide acarbose which inhibits invertase activity *in planta* resulting in an increased susceptibility of the infected plant compared to the wild type (Bonfig et al., 2010).

#### **ALKALINE/NEUTRAL INVERTASES**

A/NInvs are non-glycosylated proteins and they belong to the GH100 family (Lammens et al., 2009). They have different subcellular localizations such as cytosol, mitochondria, chloroplast and nuclei (Vargas and Salerno, 2010). A/NInvs are involved in plant growth and development, flowering and seed germination (Jia et al., 2008; Barratt et al., 2009; Welham et al., 2009). Xiang et al. (2011) demonstrated that A/NInvs are part of the antioxidant system involved in cellular reactive oxygen species homeostasis. Moreover, exogenous application of gibberellic acid (GA) rescued the delay of germination in the seeds of the A/NInv mutants suggesting a communication between A/NInv and phytohormones (Xiang et al., 2011; Martin et al., 2013).

Correlated with the increase of the CWI activity, an increase of the A/NInv activity has been observed in *Pisum sativum*, tobacco and *A. thaliana* during infection by powdery mildew (Storr and Hall, 1992), oomycetes (Essmann et al., 2008a), and the beet curly top virus (Park et al., 2013), respectively. Interestingly, in transgenic tobacco plants silenced for CWI, the A/NInv activity remained unchanged during the interaction with the oomycetic phytopathogen (Essmann et al., 2008a). The authors suggested that the CWI activity increased first and by consequence the availability of carbohydrate changes and triggers the A/NInvs activity as a secondary phenomenon in the plant immunity (Essmann et al., 2008a). By contrast, the infections of *A. thaliana* by two different nematodes *Heterodera schachtii* and *Meloidogyne javanica* led to the down-regulation of *A/NInv* gene (*AtCINV1*) reflected by a decrease of activity (Cabello et al., 2013). Thus, the importance of A/NInv might vary depending on the pathosystem.

*Abbreviations: (*+*), up-regulation; (*−*), down-regulation; (/), no change; ABA, abscisic acid; HXT, hexose transporter; PR, pathogenesis-related; ROS, reactive oxygen species; SUC, sucrose transporter; ND, not described.*

# **DEFENSE-INDUCED FEATURES AFFECTED BY SUCROSE AND INVERTASES**

#### **CLOCK, PHOTOSYNTHESIS, AND SUGAR CONTENT**

The connections between the clock, the sugars and the immunity have been previously presented (Roden and Ingle, 2009; Bolouri Moghaddam and Van Den Ende, 2013) and here we discuss the latest updates on this interconnectivity. Exogenous sucrose is able to stimulate the circadian clock by inhibiting photosynthesis and to coordinate answers during the light-dark cycles (Knight et al., 2008; Dalchau et al., 2011; Haydon et al., 2013). A new metabolic feedback loop involving the morning-expressed *pseudo response regulator 7* (*prr7*) gene was proposed by Haydon et al. (2013). At dawn, the light activates PRR7 and photosynthesis, then the photosynthetically produced derived sugars accumulate and repress the PRR7 promoter which causes the de-repression of the molecular oscillator component circadian clock associated 1 (CCA1) (Haydon et al., 2013). The clock-related genes (*cca1* and *lhy*) affect stomatal aperture after pathogen infection and suggest a crucial role of circadian clock in plant defense response (Wang et al., 2011; Zhang et al., 2013). Diurnal rhythm has been shown to regulate a CWI (LIN6) from tomato and that both CCA1 and LHY activate the *Lin6* promoter (Proels and Roitsch, 2009). During pathogen attack, the increase of CWI activity leading to an accumulation of hexoses is associated with a down-regulation of photosynthesis and expression of genes-related to photosynthesis (**Table 1**). It is noteworthy that transgenic infected plants silenced for CWI showed a delay in the reduction of photosynthesis (Kocal et al., 2008). Thus, the cross-talk between clock, sucrose and invertases tends to illustrate that a fine regulation of the sucrose/hexose ratio is crucial in defense regulation (Haydon et al., 2013).

During the day, both sucrose and starch are produced during photosynthesis. During the night, the starch, accumulated in the chloroplasts, is subsequently degraded to provide substrates for sucrose synthesis. Starch synthesis can be regulated by sucrose and clock by modulating the expression of starch synthase (Wang et al., 2001). After pathogen infection, a decrease in the starch content is observed in the infected region suggesting that the degradation of starch provides more substrates to sucrose synthesis. Interestingly, Engelsdorf et al. tested the susceptibility of starch-free *A. thaliana* mutants against biotrophic, hemibiotrophic and necrotrophic pathogens and pointed out that depending on the studied pathosystem the diurnal carbon availability is a susceptibility factor (Engelsdorf et al., 2013). Their results imply that sugar availability might impact the ability of plants to trigger defense responses.

One of the other possibilities for changing the sugar content is the regulation of the expression of the sucrose transporter. Sucrose acts on carbohydrate partitioning and phloem loading by modulating the sucrose transporter expression, such as inducing the expression of *SUT2* in tomatoes or repressing the expression of *BvSUT1* in beet (Barker et al., 2000; Vaughn et al., 2002). Depending on the stage of infection, the expression of sucrose transporters can be altered and as a consequence the sucrose partitioning can be modified. In rice infected by *Xanthomonas oryzae* pv. *Oryzae*, SWEET proteins are upregulated and sucrose accumulates in apoplast ready to be used for the pathogen growth (Chen et al., 2010, 2012). Santi et al. reported a sequential regulation of sucrose transporter genes which are first downregulated during infection of grapevine by stolbur to limit the spread and then upregulated during the recovery stage providing necessary nutrients (Santi et al., 2013a,b). It is noteworthy that during fungal infection the expression of CWI and hexose transporters displayed a correlation enhancing the hexoses supply from the phloem to the surrounding tissues during the transition from source to sink (Fotopoulos et al., 2003; Hayes et al., 2010). Moreover, Hayes et al. reported a relationship between CWI, hexose transporters and ABA biosynthesis during the transition from source to sink after infection (Hayes et al., 2010).

# **PHYTOHORMONES**

For different phytohormones such as ABA, gibberellins, ethylene and jasmonate, it was shown that they interact with the sucrose signaling pathway (Finkelstein et al., 2002; Leon and Sheen, 2003; Gibson, 2004; Heil et al., 2012). Their implication in plant defense response and the relationship with sugars have been widely discussed in various reviews (Bolouri Moghaddam and Van Den Ende, 2012, 2013).

#### **PATHOGENESIS RELATED PROTEINS**

PR proteins are synthesized in response to plant pathogen attack. Their classification and their properties have been well described (for reviews see Kitajima and Sato, 1999; Van Loon et al., 2006; Sels et al., 2008). As reported in several studies, the up-regulation of CWI due to the infection goes along with the induction of *PR* genes (**Table 1**) such as PR-1a, PR-1b, PR3, PR10, WRKY45, and NPR1 in rice (Sun et al., 2013), PR-1b and PR-Q in tobacco (Herbers et al., 1996; Schaarschmidt et al., 2007; Essmann et al., 2008b) and PR-Q, Pin-II and GluB in tomato (Kocal et al., 2008). During transgenic approaches the overexpression of CWI in tobacco or in rice presented constitutively high levels of *PR* transcripts compared to the wild type plants (Herbers et al., 1996; Sun et al., 2013). To support this idea, in different cases of infected transgenic plants silenced for CWI, the induction of *PR* genes was abolished (Schaarschmidt et al., 2007; Essmann et al., 2008b; Kocal et al., 2008). Thus CWI activity is required to enhance the expression of *PR* genes mediated by the accumulated hexoses which act as signal molecules. Besides, exogenous sucrose induced the expression of *PR* genes (Thibaud et al., 2004; Gomez-Ariza et al., 2007) confirming the idea of sucrose as an important signal molecule for plant defense response.

# **PHENYLPROPANOID PATHWAY**

The phenylalanine ammonia-lyase (PAL), a key enzyme which is involved in the phenylpropanoid pathway, leads to the biosynthesis of lignin and the production of many other important compounds such as the flavonoids, coumarins and lignans (for review see Dixon and Paiva, 1995). During infection of lupine by *Fusarium oxysporum*, sucrose induced the phenylpropanoid metabolism by stimulating the activity of PAL (Morkunas et al., 2005, 2011). Sturm and Chrispeels showed an accumulation of PAL mRNA subsequently to the increase of CWI mRNA in carrot infected by *Erwinia carotovora* (Sturm and Chrispeels, 1990). Moreover when tobacco plants are silenced for CWI, the PAL activity is delayed after infection compared with the wild type plants (Essmann et al., 2008b). Hence, the regulation of PAL is mediated by the variation of the sucrose/hexose ratio. All in all, these results demonstrate that the regulation of the expression of PAL is sugar-related.

Anthocyanin (a flavonoid) has an antimicrobial potential reducing the spread of the pathogens. The synthesis of anthocyanin is regulated by sucrose signaling pathway (Solfanelli et al., 2006) through the induction of the *PAP1/MYB75* transcription factor (Teng et al., 2005) and ABA and jasmonate pathways have a synergic effect (Loreti et al., 2008). This induction is repressed by gibberellins. At concentrations of sucrose higher than 2% the anthocyanin synthesis is induced independently of the ABA signaling pathway (Dai et al., 2014). Recently, a key positive regulator in the sucrose signaling pathway controlling the anthocyanin synthesis has been identified as the DELLA protein which targets *PAP1/MYB75* (Li et al., 2014).

In potato tubers, a transcription factor (*AN1*) was proposed to up-regulate the phenylpropanoid pathway. The authors suggested that *PAL* might be induced by *AN1* after sucrose feeding. Moreover they proposed a loop in which sucrose increases *AN1* expression while *AN1* induces sucrolytic enzymes which release hexoses used by the phenylpropanoid pathway (Payyavula et al., 2013). By the synthesis of secondary metabolites such as phenolic compounds or later on lignin, plants produce chemical and physical barriers against pathogens.

#### **CELL WALL REINFORCEMENT**

As another physical barrier, there is the deposition of callose, a β-(1,3)-glucan cell wall polymer, which is a stress related process limiting invasion by regulating the plasmodesmata and the sieve plates permeability (Chen and Kim, 2009; Luna et al., 2011). In tobacco plants overexpressing a yeast invertase in the apoplast or in the vacuole, the increase of callose deposition was comparable to that observed in wild type plants infected with potato virus Y (Herbers et al., 1996). These results were consistent with a positive regulation of callose deposition by GIF1 in rice after infection by both, bacterial and fungal pathogens (Sun et al., 2013), leading to a regulation mediated by CWI activity. Increasing concentrations of the exogenous sucrose repressed the callose deposition in *A. thaliana* cells (Luna et al., 2011) suggesting that hexose cleavage products of sucrose are responsible for the formation of the physical barrier against invading pathogens through cell wall reinforcement.

#### **CONCLUSION AND PERSPECTIVES**