Four Arabidopsis berberine bridge enzyme‐like proteins are specific oxidases that inactivate the elicitor‐active oligogalacturonides

Recognition of endogenous molecules acting as ‘damage‐associated molecular patterns’ (DAMPs) is a key feature of immunity in both animals and plants. Oligogalacturonides (OGs), i.e. fragments derived from the hydrolysis of homogalacturonan, a major component of pectin are a well known class of DAMPs that activate immunity and protect plants against several microbes. However, hyper‐accumulation of OGs severely affects growth, eventually leading to cell death and clearly pointing to OGs as players in the growth‐defence trade‐off. Here we report a mechanism that may control the homeostasis of OGs avoiding their deleterious hyper‐accumulation. By combining affinity chromatography on acrylamide‐trapped OGs and other procedures, an Arabidopsis thaliana enzyme that specifically oxidizes OGs was purified and identified. The enzyme was named OG OXIDASE 1 (OGOX1) and shown to be encoded by the gene At4g20830. As a typical flavo‐protein, OGOX1 is a sulphite‐sensitive H₂O₂‐producing enzyme that displays maximal activity on OGs with a degree of polymerization >4. OGOX1 belongs to a large gene family of mainly apoplastic putative FAD‐binding proteins [Berberine Bridge Enzyme‐like (BBE‐like); 27 members], whose biochemical and biological function is largely unexplored. We have found that at least four BBE‐like enzymes in Arabidopsis are OG oxidases (OGOX1–4). Oxidized OGs display a reduced capability of activating the immune responses and are less hydrolysable by fungal polygalacturonases. Plants overexpressing OGOX1 are more resistant to Botrytis cinerea, pointing to a crucial role of OGOX enzymes in plant immunity.     

Ethylene production in Botrytis cinerea‐ and oligogalacturonide‐induced immunity requires calcium‐dependent protein kinases

Plant immunity against pathogens is achieved through rapid activation of defense responses that occur upon sensing of microbe‐ or damage‐associated molecular patterns, respectively referred to as MAMPs and DAMPs. Oligogalacturonides (OGs), linear fragments derived from homogalacturonan hydrolysis by pathogen‐secreted cell wall‐degrading enzymes, and flg22, a 22‐amino acid peptide derived from the bacterial flagellin, represent prototypical DAMPs and MAMPs, respectively. Both types of molecules induce protection against infections. In plants, like in animals, calcium is a second messenger that mediates responses to biotic stresses by activating calcium‐binding proteins. Here we show that simultaneous loss of calcium‐dependent protein kinases CPK5, CPK6 and CPK11 affects Arabidopsis thaliana basal as well as elicitor‐ induced resistance to the necrotroph Botrytis cinerea, by affecting pathogen‐induced ethylene production and accumulation of the ethylene biosynthetic enzymes 1‐aminocyclopropane‐1‐carboxylic acid (ACC) synthase 2 (ACS2) and 6 (ACS6). Moreover, ethylene signaling contributes to OG‐triggered immunity activation, and lack of CPK5, CPK6 and CPK11 affects the duration of OG‐ and flg22‐induced gene expression, indicating that these kinases are shared elements of both DAMP and MAMP signaling pathways.     

Involvement of the glutamate receptor AtGLR3.3 in plant defense signaling and resistance to Hyaloperonospora arabidopsidis

Like their animal counterparts, plant glutamate receptor‐like (GLR) homologs are intimately associated with Ca²⁺ influx through plasma membrane and participate in various physiological processes. In pathogen‐associated molecular patterns (PAMP)‐/elicitor‐mediated resistance, Ca²⁺ fluxes are necessary for activating downstream signaling events related to plant defense. In this study, oligogalacturonides (OGs), which are endogenous elicitors derived from cell wall degradation, were used to investigate the role of Arabidopsis GLRs in defense signaling. Pharmacological investigations indicated that GLRs are partly involved in free cytosolic [Ca²⁺] ([Ca²⁺]_cyt) variations, nitric oxide (NO) production, reactive oxygen species (ROS) production and expression of defense‐related genes by OGs. In addition, wild‐type Col‐0 plants treated with the glutamate‐receptor antagonist 6,7‐dinitriquinoxaline‐2,3‐dione (DNQX) had a compromised resistance to Botrytis cinerea and Hyaloperonospora arabidopsidis. Moreover, we provide genetic evidence that AtGLR3.3 is a key component of resistance against H. arabidopsidis. In addition, some OGs‐triggered immune events such as defense gene expression, NO and ROS production are also to different extents dependent on AtGLR3.3. Taken together, these data provide evidence for the involvement of GLRs in elicitor/pathogen‐mediated plant defense signaling pathways in Arabidopsis thaliana.     

The AtrbohD-mediated oxidative burst elicited by oligogalacturonides in Arabidopsis is dispensable for the activation of defense responses effective against Botrytis cinerea

Oligogalacturonides (OGs) are endogenous elicitors of defense responses released after partial degradation of pectin in the plant cell wall. We have previously shown that, in Arabidopsis (Arabidopsis thaliana), OGs induce the expression of PHYTOALEXIN DEFICIENT3 (PAD3) and increase resistance to the necrotrophic fungal pathogen Botrytis cinerea independently of signaling pathways mediated by jasmonate, salicylic acid, and ethylene. Here, we illustrate that the rapid induction of the expression of a variety of genes by OGs is also independent of salicylic acid, ethylene, and jasmonate. OGs elicit a robust extracellular oxidative burst that is generated by the NADPH oxidase AtrbohD. This burst is not required for the expression of OG-responsive genes or for OG-induced resistance to B. cinerea, whereas callose accumulation requires a functional AtrbohD. OG-induced resistance to B. cinerea is also unaffected in powdery mildew resistant4, despite the fact that callose accumulation was almost abolished in this mutant. These results indicate that the OG-induced oxidative burst is not required for the activation of defense responses effective against B. cinerea, leaving open the question of the role of reactive oxygen species in elicitor-mediated defense.     

Functional analysis of endo‐1,4‐β‐glucanases in response to Botrytis cinerea and Pseudomonas syringae reveals their involvement in plant–pathogen interactions

Plant cell wall modification is a critical component in stress responses. Endo‐1,4‐β‐glucanases (EGs) take part in cell wall editing processes, e.g. elongation, ripening and abscission. Here we studied the infection response of Solanum lycopersicum and Arabidopsis thaliana with impaired EGs. Transgenic TomCel1 and TomCel2 tomato antisense plants challenged with Pseudomonas syringae showed higher susceptibility, callose priming and increased jasmonic acid pathway marker gene expression. These two EGs could be resistance factors and may act as negative regulators of callose deposition, probably by interfering with the defence‐signalling network. A study of a set of Arabidopsis EG T‐DNA insertion mutants challenged with P. syringae and Botrytis cinerea revealed that the lack of other EGs interferes with infection phenotype, callose deposition, expression of signalling pathway marker genes and hormonal balance. We conclude that a lack of EGs could alter plant response to pathogens by modifying the properties of the cell wall and/or interfering with signalling pathways, contributing to generate the appropriate signalling outcomes. Analysis of microarray data demonstrates that EGs are differentially expressed upon many different plant–pathogen challenges, hormone treatments and many abiotic stresses. We found some Arabidopsis EG mutants with increased tolerance to osmotic and salt stress. Our results show that impairing EGs can alter plant–pathogen interactions and may contribute to appropriate signalling outcomes in many different biotic and abiotic plant stress responses.     

Roots drive oligogalacturonide‐induced systemic immunity in tomato

Oligogalacturonides (OGs) are fragments of pectin released from the plant cell wall during insect or pathogen attack. They can be perceived by the plant as damage signals, triggering local and systemic defence responses. Here, we analyse the dynamics of local and systemic responses to OG perception in tomato roots or shoots, exploring their impact across the plant and their relevance in pathogen resistance. Targeted and untargeted metabolomics and gene expression analysis in plants treated with purified OGs revealed that local responses were transient, while distal responses were stronger and more sustained. Remarkably, changes were more conspicuous in roots, even upon foliar application of the OGs. The treatments differentially activated the synthesis of defence‐related hormones and secondary metabolites including flavonoids, alkaloids and lignans, some of them exclusively synthetized in roots. Finally, the biological relevance of the systemic defence responses activated upon OG perception was confirmed, as the treatment induced systemic resistance to Botrytis cinerea. Overall, this study shows the differential regulation of tomato defences upon OGs perception in roots and shoots and reveals the key role of roots in the coordination of the plant responses to damage sensing.     

Induced resistance to Botrytis cinerea in Capsicum annuum by a Fusarium crude elicitor fraction,free of proteins

Fusarium oxysporum f. sp. lycopersici (FOL) induces resistance in pepper against the airborne pathogen Botrytis cinerea and the soil‐borne pathogen Verticillium dahliae. However, its practical use is limited due to its pathogenicity to other crops. In this study we tested several fractions of a heat‐sterilised crude FOL‐elicitor preparation to protect pepper against B. cinerea and V. dahliae. Only the protein‐free insoluble fraction of the preparation reduced B. cinerea infection. However, none of the fractions reduce V. dahliae symptoms. The insoluble protein‐free fraction induced expression of defence genes in the plant, namely a chitinase (CACHI2), a peroxidase (CAPO1), a sesquiterpene cyclase (CASC1) and a basic PR1 (CABPR1). Even though the CASC1 gene was not induced directly after treatment with the insoluble fraction in the leaves, it was induced after B. cinerea inoculation, showing a priming effect. The insoluble protein‐free FOL‐elicitor protected pepper against the airborne pathogen through a mechanism that involves induced responses in the plant, but different to the living FOL.     

Set‐point control of RD21 protease activity by AtSerpin1 controls cell death in Arabidopsis

Programmed cell death (PCD) in plants plays a key role in defense response and is promoted by the release of compartmentalized proteases to the cytoplasm. Yet the exact identity and control of these proteases is poorly understood. Serpins are an important group of proteins that uniquely curb the activity of proteases by irreversible inhibition; however, their role in plants remains obscure. Here we show that during cell death the Arabidopsis serpin protease inhibitor, AtSerpin1, exhibits a pro‐survival function by inhibiting its target pro‐death protease, RD21. AtSerpin1 accumulates in the cytoplasm and RD21 accumulates in the vacuole and in endoplasmic reticulum bodies. Elicitors of cell death, including the salicylic acid agonist benzothiadiazole and the fungal toxin oxalic acid, stimulated changes in vacuole permeability as measured by the changes in the distribution of marker dye. Concomitantly, a covalent AtSerpin1–RD21 complex was detected indicative of a change in protease compartmentalization. Furthermore, mutant plants lacking RD21 or plants with AtSerpin1 over‐expression exhibited significantly less elicitor‐stimulated PCD than plants lacking AtSerpin1. The necrotrophic fungi Botrytis cinerea and Sclerotina sclerotiorum secrete oxalic acid as a toxin that stimulates cell death. Consistent with a pro‐death function for RD21 protease, the growth of these necrotrophs was compromised in plants lacking RD21 but accelerated in plants lacking AtSerpin1. The results indicate that AtSerpin1 controls the pro‐death function of compartmentalized protease RD21 by determining a set‐point for its activity and limiting the damage induced during cell death.     

Colonization ability as an indicator of enhanced biocontrol capacity—An example using two Bacillus amyloliquefaciens strains and Botrytis cinerea infection of tomatoes

An understanding of biocontrol activities is important when developing microorganism‐based alternatives to conventional fungicides. From our bacterial collection, we selected two strains (BBC023 and BBC047) for their outstanding antagonistic capacity against fungal phytopathogens and growth‐promoting abilities towards Arabidopsis thaliana. According to physiological and molecular characterizations, both strains were classified as Bacillus amyloliquefaciens and were tested against Botrytis cinerea in vitro and in a tomato. Both strains secrete lipopeptide‐like compounds that contribute to their in vitro antagonism. SEM‐images showed altered B. cinerea mycelial structures that were consistent with previous reports of the direct action of lipopeptides against fungal hyphae. The strains were applied to the roots (R), leaves (foliar ‐ F) or root/leaves (R/F) on tomato plants. All treatments significantly reduced the severity of B. cinerea infection (measured as a control index). However, only root applications (R and R/F) led to growth promotion in the tomato plants. We detected the production of indole acetic acid (IAA) and 2,3‐butanediol as growth promotion traits in the two strains. For both strains, the R/F treatment showed the highest control index, suggesting a synergic effect of direct antagonism against B. cinerea and resistance induction in the plant. In addition, in vitro antagonism of BBC023 and BBC047 against B. cinerea was similar; whereas in the F application, strain BBC047 significantly improved plant resistance and maintained a higher population density over time on tomato leaves, compared to BBC023. BBC047 was also able to produce a complex and robust biofilm in Msgg medium compared with that of BBC023. We linked the reduced biocontrol of BBC023 on leaves with its limited ability to generate robust biofilms and colonize the phylloplane. At last, we highlight the potential of the native Bacillus strains as promising alternatives for the development of bioproducts for sustainable agriculture.     

Pectin Biosynthesis Is Critical for Cell Wall Integrity and Immunity in Arabidopsis thaliana

Plant cell walls are important barriers against microbial pathogens. Cell walls of Arabidopsis thaliana leaves contain three major types of polysaccharides: cellulose, various hemicelluloses, and pectins. UDP-d-galacturonic acid, the key building block of pectins, is produced from the precursor UDP-d-glucuronic acid by the action of glucuronate 4-epimerases (GAEs). Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) repressed expression of GAE1 and GAE6 in Arabidopsis, and immunity to Pma ES4326 was compromised in gae6 and gae1 gae6 mutant plants. These plants had brittle leaves and cell walls of leaves had less galacturonic acid. Resistance to specific Botrytis cinerea isolates was also compromised in gae1 gae6 double mutant plants. Although oligogalacturonide (OG)-induced immune signaling was unaltered in gae1 gae6 mutant plants, immune signaling induced by a commercial pectinase, macerozyme, was reduced. Macerozyme treatment or infection with B. cinerea released less soluble uronic acid, likely reflecting fewer OGs, from gae1 gae6 cell walls than from wild-type Col-0. Although both OGs and macerozyme-induced immunity to B. cinerea in Col-0, only OGs also induced immunity in gae1 gae6. Pectin is thus an important contributor to plant immunity, and this is due at least in part to the induction of immune responses by soluble pectin, likely OGs, that are released during plant-pathogen interactions.     

The Arabidopsis LecRK‐VI.2 associates with the pattern‐recognition receptor FLS2 and primes Nicotiana benthamiana pattern‐triggered immunity

Pattern‐triggered immunity (PTI) is broad spectrum and manipulation of PTI is believed to represent an attractive way to engineer plants with broad‐spectrum disease resistance. PTI is activated upon perception of microbe‐associated molecular patterns (MAMPs) by pattern‐recognition receptors (PRRs). We have recently demonstrated that the L‐type lectin receptor kinase‐VI.2 (LecRK‐VI.2) positively regulates Arabidopsis thaliana PTI. Here we show through in vitro pull‐down, bimolecular fluorescence complementation and co‐immunoprecipitation analyses that LecRK‐VI.2 associates with the PRR FLS2. We also demonstrated that LecRK‐VI.2 from the cruciferous plant Arabidopsis remains functional after interfamily transfer to the Solanaceous plant Nicotiana benthamiana. Wild tobacco plants ectopically expressing LecRK‐VI.2 were indeed more resistant to virulent hemi‐biotrophic and necrotrophic bacteria, but not to the fungal pathogen Botrytis cinerea suggesting that, as with Arabidopsis, the LecRK‐VI.2 protective effect in N. benthamiana is bacteria specific. Ectopic expression of LecRK‐VI.2 in N. benthamiana primed PTI‐mediated reactive oxygen species production, mitogen‐activated protein kinase (MAPK) activity, callose deposition and gene expression upon treatment with the MAMP flagellin. Our findings identified LecRK‐VI.2 as a member of the FLS2 receptor complex and suggest that heterologous expression of components of PRR complexes can be used as tools to engineer plant disease resistance to bacteria.     

IDL6‐HAE/HSL2 impacts pectin degradation and resistance to Pseudomonas syringae pv tomato DC3000 in Arabidopsis leaves

Plant cell walls undergo dynamic structural and chemical changes during plant development and growth. Floral organ abscission and lateral root emergence are both accompanied by cell‐wall remodeling, which involves the INFLORESCENCE DEFICIENT IN ABSCISSION (IDA)‐derived peptide and its receptors, HAESA (HAE) and HAESA‐LIKE2 (HSL2). Plant cell walls also act as barriers against pathogenic invaders. Thus, the cell‐wall remodeling during plant development could have an influence on plant resistance to phytopathogens. Here, we identified IDA‐like 6 (IDL6), a gene that is prominently expressed in Arabidopsis leaves. IDL6 expression in Arabidopsis leaves is significantly upregulated when the plant is suffering from attacks of the bacterial Pseudomonas syringae pv. tomato (Pst) DC3000. IDL6 overexpression and knockdown lines respectively decrease and increase the Arabidopsis resistance to Pst DC3000, indicating that the gene promotes the Arabidopsis susceptibility to Pst DC3000. Moreover, IDL6 promotes the expression of a polygalacturonase (PG) gene, ADPG2, and increases PG activity in Arabidopsis leaves, which in turn reduces leaf pectin content and leaf robustness. ADPG2 overexpression restrains Arabidopsis resistance to Pst DC3000, whereas ADPG2 loss‐of‐function mutants increase the resistance to the bacterium. Pst DC3000 infection elevates the ADPG2 expression partially through HAE and HSL2. Taken together, our results suggest that IDL6‐HAE/HSL2 facilitates the ingress of Pst DC3000 by promoting pectin degradation in Arabidopsis leaves, and Pst DC3000 might enhance its infection by manipulating the IDL6‐HAE/HSL2‐ADPG2 signaling pathway.     

Resistance to Botrytis cinerea induced in Arabidopsis by elicitors is independent of salicylic acid, ethylene, or jasmonate signaling but requires PHYTOALEXIN DEFICIENT3

Oligogalacturonides (OGs) released from plant cell walls by pathogen polygalacturonases induce a variety of host defense responses. Here we show that in Arabidopsis (Arabidopsis thaliana), OGs increase resistance to the necrotrophic fungal pathogen Botrytis cinerea independently of jasmonate (JA)-, salicylic acid (SA)-, and ethylene (ET)-mediated signaling. Microarray analysis showed that about 50% of the genes regulated by OGs, including genes encoding enzymes involved in secondary metabolism, show a similar change of expression during B. cinerea infection. In particular, expression of PHYTOALEXIN DEFICIENT3 (PAD3) is strongly up-regulated by both OGs and infection independently of SA, JA, and ET. OG treatments do not enhance resistance to B. cinerea in the pad3 mutant or in underinducer after pathogen and stress1, a mutant with severely impaired PAD3 expression in response to OGs. Similarly to OGs, the bacterial flagellin peptide elicitor flg22 also enhanced resistance to B. cinerea in a PAD3-dependent manner, independently of SA, JA, and ET. This work suggests, therefore, that elicitors released from the cell wall during pathogen infection contribute to basal resistance against fungal pathogens through a signaling pathway also activated by pathogen-associated molecular pattern molecules.     

Introduction of chemically labile substructures into Arabidopsis lignin through the use of LigD,the Cα‐dehydrogenase from Sphingobium sp. strain SYK‐6

Bacteria‐derived enzymes that can modify specific lignin substructures are potential targets to engineer plants for better biomass processability. The Gram‐negative bacterium Sphingobium sp. SYK‐6 possesses a Cα‐dehydrogenase (LigD) enzyme that has been shown to oxidize the α‐hydroxy functionalities in β–O–4‐linked dimers into α‐keto analogues that are more chemically labile. Here, we show that recombinant LigD can oxidize an even wider range of β–O–4‐linked dimers and oligomers, including the genuine dilignols, guaiacylglycerol‐β‐coniferyl alcohol ether and syringylglycerol‐β‐sinapyl alcohol ether. We explored the possibility of using LigD for biosynthetically engineering lignin by expressing the codon‐optimized ligD gene in Arabidopsis thaliana. The ligD cDNA, with or without a signal peptide for apoplast targeting, has been successfully expressed, and LigD activity could be detected in the extracts of the transgenic plants. UPLC‐MS/MS‐based metabolite profiling indicated that levels of oxidized guaiacyl (G) β–O–4‐coupled dilignols and analogues were significantly elevated in the LigD transgenic plants regardless of the signal peptide attachment to LigD. In parallel, 2D NMR analysis revealed a 2.1‐ to 2.8‐fold increased level of G‐type α‐keto‐β–O–4 linkages in cellulolytic enzyme lignins isolated from the stem cell walls of the LigD transgenic plants, indicating that the transformation was capable of altering lignin structure in the desired manner.     

The Antimicrobial Peptide Thanatin Reduces Fungal Infections in Arabidopsis

We explored the antifungal activity of thanatin, a 21 amino acid synthetic peptide from the hemipteran spined soldier bug Podisus maculiventris, against the mycotoxin‐producing plant pathogenic ascomycete Fusarium graminearum. In vitro germination assays showed complete inhibition of macroconidia germination and mycelia growth by >10 μm thanatin. Moreover, detached leaves of thanatin‐expressing Arabidopsis thaliana plants displayed enhanced resistance towards colonization with F. graminearum. Consistent with this, the plants showed also enhanced resistance of detached leaves to colonization with Botrytis cinerea. The results demonstrate a potential of thanatin for use in plant protection.     

Silencing of OPR3 in tomato reveals the role of OPDA in callose deposition during the activation of defense responses against Botrytis cinerea

Cis‐(+)‐12‐oxo‐phytodienoic acid (OPDA) is likely to play signaling roles in plant defense that do not depend on its further conversion to the phytohormone jasmonic acid. To elucidate the role of OPDA in Solanum lycopersicum (tomato) plant defense, we have silenced the 12‐oxophytodienoate reductase 3 (OPR3) gene. Two independent transgenic tomato lines (SiOPR3‐1 and SiOPR3‐2) showed significantly reduced OPR3 expression upon infection with the necrotrophic pathogen Botrytis cinerea. Moreover, SiOPR3 plants are more susceptible to this pathogen, and this susceptibility is accompanied by a significant decrease in OPDA levels and by the production of JA‐Ile being almost abolished. OPR3 silencing also leads to a major reduction in the expression of other genes of the jasmonic acid (JA) synthesis and signaling pathways after infection. These results confirm that in tomato plants, as in Arabidopsis, OPR3 determines OPDA availability for JA biosynthesis. In addition, we show that an intact JA biosynthetic pathway is required for proper callose deposition, as its pathogen‐induced accumulation is reduced in SiOPR3 plants. Interestingly, OPDA, but not JA, treatment restored basal resistance to B. cinerea and induced callose deposition in SiOPR3‐1 and SiOPR3‐2 transgenic plants. These results provide clear evidence that OPDA by itself plays a major role in the basal defense of tomato plants against this necrotrophic pathogen.