Infection of Arabidopsis with a necrotrophic pathogen,Botrytis cinerea,elicits various defense responses but does not induce systemic acquired resistance (SAR)

Botrytis cinerea is a non-specific necrotrophic pathogen that attacks more than 200 plant species. In contrast to biotrophs, the necrotrophs obtain their nutrients by first killing the host cells. Many studies have shown that infection of plants by necrosis-causing pathogens induces a systemic acquired resistance (SAR), which provides protection against successive infections by a range of pathogenic organisms. We analyzed the role of SAR in B. cinerea infection of Arabidopsis. We show that although B. cinerea induced necrotic lesions and camalexin biosynthesis, it did not induce SAR-mediated protection against virulent strains of Pseudomonas syringae, or against subsequent B. cinerea infections. Induction of SAR with avirulent P. syringae or by chemical treatment with salicylic acid (SA) or benzothiadiazole also failed to inhibit B. cinerea growth, although removal of basal SA accumulation by expression of a bacterial salicylate hydroxylase (NahG) gene or by infiltration of 2-aminoindan-2-phosphonic acid, an inhibitor of phenylpropanoid pathway, increased B. cinerea disease symptoms. In addition, we show that B. cinerea induced expression of genes associated with SAR, general stress and ethylene/jasmonate-mediated defense pathways. Thus, B. cinerea does not induce SAR nor is it affected by SAR, making it a rare example of a necrogenic pathogen that does not cause SAR.     

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.     

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.     

The role of phytohormones in basal resistance and Trichoderma-induced systemic resistance to Botrytis cinerea in Arabidopsis thaliana

Thirty-six phytohormone-affected mutants of Arabidopsis thaliana (L.) Heynh. and their parental ecotypes were tested for resistance/susceptibility to Botrytis cinerea Pers.; Fr. and ability to develop Trichoderma-mediated induced systemic resistance (ISR). Ecotype Colombia-0 (Col-0) was relatively resistant to B. cinerea, and Trichoderma harzianum Rifai T39 application at sites spatially separated (roots) from the B. cinerea inoculation (leaves) resulted in reduction of grey mold symptoms. Ecotypes Wassilewskija-4, Nossen-0 and Landsberg-0 had low levels of basal resistance to B. cinerea and were unable to express ISR. Mutants derived from ISR-non-inducible ecotypes displayed ISR-non-inducible phenotypes, whereas the ISR inducibility of mutants derived from the ISR-inducible genotype Col-0 varied according to the type of mutant. Thus, salicylic acid (SA)-impaired mutants derived from Col-0 were ISR-inducible, while ethylene/jasmonic acid (ethylene/JA)-impaired mutants of the same origin were ISR-non-inducible. SA-impaired mutants retained basal level of resistance to B. cinerea, while most ethylene/JA-impaired mutants were highly susceptible. Abscisic acid- and gibberellin-impaired mutants were highly susceptible to B. cinerea and showed ISR-non-inducible phenotypes irrespective of their lines of origin. Auxin-resistant mutants derived from Col-0 were ISR-inducible; mutant originating from Landsberg-0 and mutants which were resistant to both auxin and ethylene were ISR-non-inducible. Most of the arabidopsis genotypes which were unable to express Trichoderma-mediated ISR against B. cinerea exhibited enhanced susceptibility to this pathogen. T. harzianum treatments enhanced the growth of arabidopsis plants regardless of genotype or ISR inducibility.     

A non-JA producing oxophytodienoate reductase functions in salicylic acid-mediated antagonism with jasmonic acid during pathogen attack

Peroxisome-localized oxo-phytodienoic acid (OPDA) reductases (OPR) are enzymes converting 12-OPDA into jasmonic acid (JA). However, the biochemical and physiological functions of the cytoplasmic non-JA producing OPRs remain largely unknown. Here, we generated Mutator-insertional mutants of the maize OPR2 gene and tested its role in resistance to pathogens with distinct lifestyles. Functional analyses showed that the opr2 mutants were more susceptible to the (hemi)biotrophic pathogens Colletotrichum graminicola and Ustilago maydis, but were more resistant to the necrotrophic fungus Cochliobolus heterostrophus. Hormone profiling revealed that increased susceptibility to C. graminicola was associated with decreased salicylic acid (SA) but increased JA levels. Mutation of the JA-producing lipoxygenase 10 (LOX10) reversed this phenotype in the opr2 mutant background, corroborating the notion that JA promotes susceptibility to this pathogen. Exogenous SA did not rescue normal resistance levels in opr2 mutants, suggesting that this SA-inducible gene is the key downstream component of the SA-mediated defences against C. graminicola. Disease assays of the single and double opr2 and lox10 mutants and the JA-deficient opr7opr8 mutants showed that OPR2 negatively regulates JA biosynthesis, and that JA is required for resistance against C. heterostrophus. Overall, this study uncovers a novel function of a non-JA producing OPR as a major negative regulator of JA biosynthesis during pathogen infection, a function that leads to its contrasting contribution to either resistance or susceptibility depending on pathogen lifestyle.     

Cyclic lipopeptides from Bacillus subtilis activate distinct patterns of defence responses in grapevine

Non‐self‐recognition of microorganisms partly relies on the perception of microbe‐associated molecular patterns (MAMPs) and leads to the activation of an innate immune response. Bacillus subtilis produces three main families of cyclic lipopeptides (LPs), namely surfactins, iturins and fengycins. Although LPs are involved in induced systemic resistance (ISR) activation, little is known about defence responses induced by these molecules and their involvement in local resistance to fungi. Here, we showed that purified surfactin, mycosubtilin (iturin family) and plipastatin (fengycin family) are perceived by grapevine plant cells. Although surfactin and mycosubtilin stimulated grapevine innate immune responses, they differentially activated early signalling pathways and defence gene expression. By contrast, plipastatin perception by grapevine cells only resulted in early signalling activation. Gene expression analysis suggested that mycosubtilin activated salicylic acid (SA) and jasmonic acid (JA) signalling pathways, whereas surfactin mainly induced an SA‐regulated response. Although mycosubtilin and plipastatin displayed direct antifungal activity, only surfactin and mycosubtilin treatments resulted in a local long‐lasting enhanced tolerance to the necrotrophic fungus Botrytis cinerea in grapevine leaves. Moreover, challenge with specific strains overproducing surfactin and mycosubtilin led to a slightly enhanced stimulation of the defence response compared with the LP‐non‐producing strain of B. subtilis. Altogether, our results provide the first comprehensive view of the involvement of LPs from B. subtilis in grapevine plant defence and local resistance against the necrotrophic pathogen Bo. cinerea. Moreover, this work is the first to highlight the ability of mycosubtilin to trigger an immune response in plants.     

A mutation in the expansin‐like A2 gene enhances resistance to necrotrophic fungi and hypersensitivity to abiotic stress in Arabidopsis thaliana

Expansins are cell wall loosening agents, known for their endogenous function in cell wall extensibility. The Arabidopsis expansin‐like A2 (EXLA2) gene was identified by its down‐regulation in response to infection by the necrotrophic pathogen Botrytis cinerea, and by the reduced susceptibility of an exla2 mutant to the same pathogen. The exla2 mutant was equally susceptible to Pseudomonas syringae pv. tomato, but was more resistant to the necrotrophic fungus Alternaria brassicicola, when compared with the wild‐type or with transgenic, ectopic EXLA2‐overexpressing lines. The exla2 mutants also enhanced tolerance to the phytoprostane‐A₁. This suggests that the absence or down‐regulation of EXLA2 leads to increased resistance to B. cinerea in a CORONATINE INSENSITIVE 1 (COI1)‐dependent manner, and this down‐regulation can be achieved by phytoprostane‐A₁ treatment. EXLA2 is induced significantly by salinity and cold, and by the exogenous application of abscisic acid. The exla2 mutant also showed hypersensitivity towards increased salt and cold, and this hypersensitivity required a functional abscisic acid pathway. The differential temporal expression of EXLA2 and the phenotypes in transgenic plants with altered expression of EXLA2 indicate that plant cell wall structure is an important player during Arabidopsis developmental stages. Our results indicate that EXLA2 appears to be important in response to various biotic and abiotic stresses, particularly in the pathogenesis of necrotrophic pathogens and in the tolerance to abiotic stress.     

Overexpressing the N-terminus of CATALASE2 enhances plant jasmonic acid biosynthesis and resistance to necrotrophic pathogen Botrytis cinerea B05.10

Salicylic acid (SA) acts antagonistically to jasmonic acid (JA) in plant immunity. We previously reported that CATALASE2 (CAT2) promotes JA-biosynthetic acyl-CoA oxidase (ACX) activity to enhance plant resistance to necrotrophic Botrytis cinerea, and SA represses JA biosynthesis through inhibiting CAT2 activity, while the underlying mechanism remains to be further elucidated. Here, we report that the truncated CAT2 N-terminus (CAT2-N) interacts with and promotes ACX2/3, and CAT2-N-overexpressing plants have increased JA accumulation and enhanced resistance to B. cinerea B05.10, but compromised antagonism of SA on JA. Catalase inhibitor treatment or mutating CAT2 active amino acids abolished CAT2 H₂O₂-decomposing activity but did not affect its promotion of ACX2/3 activity via interaction. CAT2-N, a truncated protein with no catalase activity, interacted with and promoted ACX2/3. Overexpressing CAT2-N in Arabidopsis plants resulted in increased ACX activity, higher JA accumulation, and stronger resistance to B. cinerea B05.10 infection. Additionally, SA dramatically repressed JA biosynthesis and resistance to B. cinerea in the wild type but not in the CAT2-N-overexpressing plants. Together, our study reveals that CAT2-N can be utilized as an accelerator for JA biosynthesis during plant resistance to B. cinerea B05.10, and this truncated protein partly relieves SA repression of JA biosynthesis in plant defence responses.     

Characterization of Botrytis–plant interactions using PathTrack©—an automated system for dynamic analysis of disease development

The measurement of disease development is integral in studies on plant–microbe interactions. To address the need for a dynamic and quantitative disease evaluation, we developed PathTrack^©, and used it to analyse the interaction of plants with Botrytis cinerea. PathTrack^© is composed of an infection chamber, a photography unit and software that produces video files and numerical values of disease progression. We identified a previously unrecognized infection stage and determined numerical parameters of pathogenic development. Using these parameters, we identified differences in disease dynamics between seemingly similar B. cinerea pathogenicity mutants, and revealed new details on plant susceptibility to the fungus. We showed that the difference between the lesion expansion rate on leaves and colony spreading rate on artificial medium reflects the levels of the plant immune system, suggesting that this parameter can be used to quantify plant defence. Our results shed new light and reveal new details of the interaction between the model necrotrophic pathogen B. cinerea and plants. The concept that we present is universal and may be applied to facilitate the study of various types of plant–pathogen association.     

Priming of the Arabidopsis pattern‐triggered immunity response upon infection by necrotrophic Pectobacterium carotovorum bacteria

Boosted responsiveness of plant cells to stress at the onset of pathogen‐ or chemically induced resistance is called priming. The chemical β‐aminobutyric acid (BABA) enhances Arabidopsis thaliana resistance to hemibiotrophic bacteria through the priming of the salicylic acid (SA) defence response. Whether BABA increases Arabidopsis resistance to the necrotrophic bacterium Pectobacterium carotovorum ssp. carotovorum (Pcc) is not clear. In this work, we show that treatment with BABA protects Arabidopsis against the soft‐rot pathogen Pcc. BABA did not prime the expression of the jasmonate/ethylene‐responsive gene PLANT DEFENSIN 1.2 (PDF1.2), the up‐regulation of which is usually associated with resistance to necrotrophic pathogens. Expression of the SA marker gene PATHOGENESIS RELATED 1 (PR1) on Pcc infection was primed by BABA treatment, but SA‐defective mutants demonstrated a wild‐type level of BABA‐induced resistance against Pcc. BABA primed the expression of the pattern‐triggered immunity (PTI)‐responsive genes FLG22‐INDUCED RECEPTOR‐LIKE KINASE 1 (FRK1), ARABIDOPSIS NON‐RACE SPECIFIC DISEASE RESISTANCE GENE (NDR1)/HAIRPIN‐INDUCED GENE (HIN1)‐LIKE 10 (NHL10) and CYTOCHROME P450, FAMILY 81 (CYP81F2) after inoculation with Pcc or after treatment with purified bacterial microbe‐associated molecular patterns, such as flg22 or elf26. PTI‐mediated callose deposition was also potentiated in BABA‐treated Arabidopsis, and BABA boosted Arabidopsis stomatal immunity to Pcc. BABA treatment primed the PTI response in the SA‐defective mutants SA induction deficient 2‐1 (sid2‐1) and phytoalexin deficient 4‐1 (pad4‐1). In addition, BABA priming was associated with open chromatin configurations in the promoter region of PTI marker genes. Our data indicate that BABA primes the PTI response upon necrotrophic bacterial infection and suggest a role for the PTI response in BABA‐induced resistance.     

The Botrytis cinerea cerato‐platanin BcSpl1 is a potent inducer of systemic acquired resistance (SAR) in tobacco and generates a wave of salicylic acid expanding from the site of application

Systemic acquired resistance (SAR) is a potent plant defence system that, in response to a first contact with a plant pathogen, prepares the whole plant for subsequent attacks, so that it becomes more resistant to the same and to other pathogens. BcSpl1, a cerato‐platanin family protein abundantly secreted by Botrytis cinerea, is required for full virulence and elicits the hypersensitive response in the host. Here, we report that BcSpl1 is also able to induce in tobacco systemic resistance to two plant pathogens, Pseudomonas syringae and B. cinerea, which correlates with the induction of two pathogenesis‐related genes, PR‐1a and PR‐5. Levels of salicylic acid were quantified in situ on BcSpl1 infiltration, and a wave of salicylic acid departing from the point of infiltration and running through the leaf was observed, as well as the appearance of this plant hormone in the neighbouring leaves as early as 3 days after infiltration.     

The Elongator complex‐associated protein DRL1 plays a positive role in immune responses against necrotrophic fungal pathogens in Arabidopsis

DEFORMED ROOT AND LEAVES1 (DRL1) is an Arabidopsis homologue of the yeast TOXIN TARGET4 (TOT4)/KILLER TOXIN‐INSENSITIVE12 (KTI12) protein that is physically associated with the RNA polymerase II‐interacting protein complex named Elongator. Mutations in DRL1 and Elongator lead to similar morphological and molecular phenotypes, suggesting that DRL1 and Elongator may functionally overlap in Arabidopsis. We have shown previously that Elongator plays an important role in both salicylic acid (SA)‐ and jasmonic acid (JA)/ethylene (ET)‐mediated defence responses. Here, we tested whether DRL1 also plays a similar role as Elongator in plant immune responses. Our results show that, although DRL1 partially contributes to SA‐induced cytotoxicity, it does not play a significant role in SA‐mediated expression of PATHOGENESIS‐RELATED genes and resistance to the virulent bacterial pathogen Pseudomonas syringae pv. maculicola ES4326. In contrast, DRL1 is required for JA/ET‐ and necrotrophic fungal pathogen Botrytis cinerea‐induced defence gene expression and for resistance to B. cinerea and Alternaria brassicicola. Furthermore, unlike the TOT4/KTI12 gene which, when overexpressed in yeast, confers zymocin resistance, a phenotype of the tot4/kti12 mutant, overexpression of DRL1 does not change B. cinerea‐induced defence gene expression and resistance to this pathogen. Finally, DRL1 contains an N‐terminal P‐loop and a C‐terminal calmodulin (CaM)‐binding domain and is a CaM‐binding protein. We demonstrate that both the P‐loop and the CaM‐binding domain are essential for the function of DRL1 in B. cinerea‐induced expression of PDF1.2 and ORA59, and in resistance to B. cinerea, suggesting that the function of DRL1 in plant immunity may be regulated by ATP/GTP and CaM binding.     

Functional analysis of an extracellular catalase of Botrytis cinerea

There is evidence that the necrotrophic fungal pathogen Botrytis cinerea is exposed to oxidative processes within plant tissues. The pathogen itself also generates active oxygen species and H₂O₂ as pathogenicity factors. Our aim was to study how the pathogen may defend itself against cellular damage caused by the accumulation of H₂O₂ and the role of an extracellular catalase in its detoxification during the infection of tomato and bean plants by B. cinerea. Chloronaphthol staining followed by light microscopy showed that H₂O₂ accumulates in the infection zone in tomato and bean leaves. An extracellular catalase gene (denominated Bccat2) was cloned from B. cinerea. Exposure of mycelium to H₂O₂ in liquid culture resulted in increased Bccat2 mRNA levels in a concentration-dependent manner. Bccat2 mRNA was detected at early stages of tomato leaf infection, suggesting that B. cinerea experiences oxidative stress. Bccat2-deficient mutants were generated by transformation-mediated gene disruption. Mutants were more sensitive then the wild-type strain to H₂O₂in vitro, but they partly compensated for the absence of BcCAT2 by activating other protective mechanisms in the presence of H₂O₂. Bccat2-deficient mutants did not display a consistent reduction of virulence on bean and tomato leaves. Cerium chloride staining of infected leaf tissue for ultrastructural studies showed that Bccat2-deficient mutants were exposed to H₂O₂ comparably to the wild-type. The results suggest that B. cinerea is a robust pathogen adapted to growing in hostile oxidizing environments in host tissues.     

Activation of Plant Innate Immunity by Extracellular High Mobility Group Box 3 and Its Inhibition by Salicylic Acid

Damage-associated molecular pattern molecules (DAMPs) signal the presence of tissue damage to induce immune responses in plants and animals. Here, we report that High Mobility Group Box 3 (HMGB3) is a novel plant DAMP. Extracellular HMGB3, through receptor-like kinases BAK1 and BKK1, induced hallmark innate immune responses, including i) MAPK activation, ii) defense-related gene expression, iii) callose deposition, and iv) enhanced resistance to Botrytis cinerea. Infection by necrotrophic B. cinerea released HMGB3 into the extracellular space (apoplast). Silencing HMGBs enhanced susceptibility to B. cinerea, while HMGB3 injection into apoplast restored resistance. Like its human counterpart, HMGB3 binds salicylic acid (SA), which results in inhibition of its DAMP activity. An SA-binding site mutant of HMGB3 retained its DAMP activity, which was no longer inhibited by SA, consistent with its reduced SA-binding activity. These results provide cross-kingdom evidence that HMGB proteins function as DAMPs and that SA is their conserved inhibitor.