Characterization of Defense Signaling Pathways of <Emphasis Type="Italic">Brassica napus</Emphasis> and <Emphasis Type="Italic">Brassica carinata</Emphasis> in Response to <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> Challenge

Canola (Brassica napus L.) is an agriculturally and economically important crop in Canada, and its growth and yield are frequently influenced by fungal pathogens. Sclerotinia sclerotiorum is among those fungal pathogens and causes stem rot disease in B. napus whereas it has been reported that Brassica carinata is moderately tolerant to S. sclerotiorum. Jasmonic acid/ethylene (JA/ET) and salicylic acid (SA) are phytohormones that are known to be involved in plant disease responses. To investigate the defense signaling cascades involved in the interaction of B. napus and B. carinata with S. sclerotiorum, we examined the expression of five orthologs of B. napus genes involved in JA/ET or SA signaling pathways using quantitative RT-PCR. Our results indicated that there are differences in the timing of JA/ET and SA signaling pathways between B. napus and B. carinata. Our results in these two Brassica species also support previous observations that necrotrophic pathogens trigger JA/ET signaling in response to infection. Finally, we observed that transgenic canola expressing 1-aminocyclopropane-1-carboxylate-deaminase producing low levels of ET was relatively more susceptible to S. sclerotiorum than its wild-type counterpart, suggesting that ET inhibits S. sclerotiorum-induced symptom development.     

The eukaryotic protein kinase superfamily of the necrotrophic fungal plant pathogen,Sclerotinia sclerotiorum

Protein kinases have been implicated in the regulation of many processes that guide pathogen development throughout the course of infection. A survey of the Sclerotinia sclerotiorum genome for genes encoding proteins containing the highly conserved eukaryotic protein kinase (ePK) domain, the largest protein kinase superfamily, revealed 92 S. sclerotiorum ePKs. This review examines the composition of the S. sclerotiorum ePKs based on conserved motifs within the ePK domain family, and relates this to orthologues found in other filamentous fungi and yeasts. The ePKs are also discussed in terms of their proposed role(s) in aspects of host pathogenesis, including the coordination of mycelial growth/development and deployment of pathogenicity determinants in response to environmental stimuli, nutrients and stress.     

Arabidopsis GDSL1 overexpression enhances rapeseed Sclerotinia sclerotiorum resistance and the functional identification of its homolog in Brassica napus

Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum is a devastating disease of rapeseed (Brassica napus L.). To date, the genetic mechanisms of rapeseed’ interactions with S. sclerotiorum are not fully understood, and molecular‐based breeding is still the most effective control strategy for this disease. Here, Arabidopsis thaliana GDSL1 was characterized as an extracellular GDSL lipase gene functioning in Sclerotinia resistance. Loss of AtGDSL1 function resulted in enhanced susceptibility to S. sclerotiorum. Conversely, overexpression of AtGDSL1 in B. napus enhanced resistance, which was associated with increased reactive oxygen species (ROS) and salicylic acid (SA) levels, and reduced jasmonic acid levels. In addition, AtGDSL1 can cause an increase in lipid precursor phosphatidic acid levels, which may lead to the activation of downstream ROS/SA defence‐related pathways. However, the rapeseed BnGDSL1 with highest sequence similarity to AtGDSL1 had no effect on SSR resistance. A candidate gene association study revealed that only one AtGDSL1 homolog from rapeseed, BnaC07g35650D (BnGLIP1), significantly contributed to resistance traits in a natural B. napus population, and the resistance function was also confirmed by a transient expression assay in tobacco leaves. Moreover, genomic analyses revealed that BnGLIP1 locus was embedded in a selected region associated with SSR resistance during the breeding process, and its elite allele type belonged to a minor allele in the population. Thus, BnGLIP1 is the functional equivalent of AtGDSL1 and has a broad application in rapeseed S. sclerotiorum‐resistance breeding.     

Differentially Expressed Proteins and Associated Histological and Disease Progression Changes in Cotyledon Tissue of a Resistant and Susceptible Genotype of Brassica napus Infected with Sclerotinia sclerotiorum

Sclerotinia rot caused by Sclerotinia sclerotiorum is one of the most serious diseases of oilseed rape. To understand the resistance mechanisms in the Brassica napus to S. sclerotiorum, comparative disease progression, histological and proteomic studies were conducted of two B. napus genotypes (resistant cv. Charlton, susceptible cv. RQ001-02M2). At 72 and 96 h post inoculation (hpi), lesion size on cotyledons was significantly (P≤0.001) smaller in the resistant Charlton. Anatomical investigations revealed impeded fungal growth (at 24 hpi and onwards) and hyphal disintegration only on resistant Charlton. Temporal changes (12, 24, 48 and 72 hpi) in protein profile showed certain enzymes up-regulated only in resistant Charlton, such as those related to primary metabolic pathways, antioxidant defence, ethylene biosynthesis, pathogenesis related proteins, protein synthesis and protein folding, play a role in mediating defence responses against S. sclerotiorum. Similarly a eukaryotic translation initiation factor 5A enzyme with increased abundance in susceptible RQ001-02M2 and decreased levels in resistant Charlton has a role in increased susceptibility to this pathogen. This is the first time that the expression of these enzymes has been shown to be associated with mediating the defence response against S. sclerotinia in cotyledon tissue of a resistant cultivar of B. napus at a proteomics level. This study not only provides important new insights into the resistance mechanisms within B. napus against S. sclerotiorum, but opens the way for novel engineering of new B. napus varieties that over-express these key enzymes as a strategy to enhance resistance and better manage this devastating pathogen.     

The broad host range pathogen Sclerotinia sclerotiorum produces multiple effector proteins that induce host cell death intracellularly

Sclerotinia sclerotiorum is a broad host range necrotrophic fungal pathogen, which causes disease on many economically important crop species. S. sclerotiorum has been shown to secrete small effector proteins to kill host cells and acquire nutrients. We set out to discover novel necrosis-inducing effectors and characterize their activity using transient expression in Nicotiana benthamiana leaves. Five intracellular necrosis-inducing effectors were identified with differing host subcellular localization patterns, which were named intracellular necrosis-inducing effector 1–5 (SsINE1–5). We show for the first time a broad host range pathogen effector, SsINE1, that uses an RxLR-like motif to enter host cells. Furthermore, we provide preliminary evidence that SsINE5 induces necrosis via an NLR protein. All five of the identified effectors are highly conserved in globally sourced S. sclerotiorum isolates. Taken together, these results advance our understanding of the virulence mechanisms employed by S. sclerotiorum and reveal potential avenues for enhancing genetic resistance to this damaging fungal pathogen.     

Transgenic canola lines expressing pea defense gene DRR206 have resistance to aggressive blackleg isolates and to Rhizoctonia solani

We have previously demonstrated that transgenic Brassica napus plants expressing pea DRR206 constitutively are resistant to the hemibiotrophic blackleg fungus, Leptosphaeria maculans isolate PG2. The present work seeks to determine whether DRR206 is effective against a wider range of fungi. Transgenic plants expressing DRR206 exhibit decreased severity of stem canker in adult plants inoculated with aggressive L. maculans isolates PG3 and PG4. Decreased seedling mortality with the biotrophic root pathogen Rhizoctonia solani is also seen. Finally, leaves of DRR206 transgenic plants inoculated with the necrotroph Sclerotinia sclerotiorum show smaller lesions at 48 h after inoculation, leading to a delay, but not a prevention, of disease development. These results demonstrate the effectiveness of DRR206 against several fungal species with three distinct modes of pathogenicity. Although its precise function remains to be determined, a recent report shows that pea DRR206 shares strong amino acid sequence similarity with `dirigent proteins' which couple monolignol radicals to form the lignan (+) pinoresinol.     

ANGUSTIFOLIA negatively regulates resistance to Sclerotinia sclerotiorum via modulation of PTI and JA signalling pathways in Arabidopsis thaliana

Sclerotinia sclerotiorum is a devastating pathogen that infects a broad range of host plants. The mechanism underlying plant defence against fungal invasion is still not well characterized. Here, we report that ANGUSTIFOLIA (AN), a CtBP family member, plays a role in the defence against S. sclerotiorum attack. Arabidopsis an mutants exhibited stronger resistance to S. sclerotiorum at the early stage of infection than wild-type plants. Accordingly, an mutants exhibited stronger activation of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) responses, including mitogen-activated protein kinase activation, reactive oxygen species accumulation, callose deposition, and the expression of PTI-responsive genes, upon treatment with PAMPs/microbe-associated molecular patterns. Moreover, Arabidopsis lines overexpressing AN were more susceptible to S. sclerotiorum and showed defective PTI responses. Our luminometry, bimolecular fluorescence complementation, coimmunoprecipitation, and in vitro pull-down assays indicate that AN interacts with allene oxide cyclases (AOC), essential enzymes involved in jasmonic acid (JA) biosynthesis, negatively regulating JA biosynthesis in response to S. sclerotiorum infection. This work reveals AN is a negative regulator of the AOC-mediated JA signalling pathway and PTI activation.     

LIK1, A CERK1-Interacting Kinase,Regulates Plant Immune Responses in Arabidopsis

Chitin, an integral component of the fungal cell wall, is one of the best-studied microbe-associated molecular patterns. Previous work identified a LysM receptor-like kinase (LysM-RLK1/CERK1) as the primary chitin receptor in Arabidopsis. In order to identify proteins that interact with CERK1, we conducted a yeast two-hybrid screen using the intracellular kinase domain of CERK1 as the bait. This screen identified 54 putative CERK1-interactors. Screening mutants defective in 43 of these interacting proteins identified only two, a calmodulin like protein (At3g10190) and a leucine-rich repeat receptor like kinase (At3g14840), which differed in their response to pathogen challenge. In the present work, we focused on characterizing the LRR-RLK gene where mutations altered responses to chitin elicitation. This LRR-RLK was named LysM RLK1-interacting kinase 1 (LIK1). The interaction between CERK1 and LIK1 was confirmed by co-immunoprecipitation using protoplasts and transgenic plants. In vitro experiments showed that LIK1 was directly phosphorylated by CERK1. In vivo phosphorylation assays showed that Col-0 wild-type plants have more phosphorylated LIK1 than cerk1 mutant plants, suggesting that LIK1 may be directly phosphorylated by CERK1. Lik1 mutant plants showed an enhanced response to both chitin and flagellin elicitors. In comparison to the wild-type plants, lik1 mutant plants were more resistant to the hemibiotrophic pathogen Pseudomonas syringae, but more susceptible to the necrotrophic pathogen Sclerotinia sclerotiorum. Consistent with the enhanced susceptibility to necrotrophs, lik1 mutants showed reduced expression of genes involved in jasmonic acid and ethylene signaling pathways. These data suggest that LIK1 directly interacts with CERK1 and regulates MAMP-triggered innate immunity.     

Differential expression of duplicated peroxidase genes in the allotetraploid Brassica napus

Gene redundancy due to polyploidization provides a selective advantage for plant adaptation. We examined the expression patterns of two peroxidase genes (BnPOX1 and BnPOX2) in the natural allotetraploid Brassica napus and the model diploid progenitors Brassica rapa (Br) and Brassica oleracea (Bo) in response to the fungal pathogen Sclerotinia sclerotiorum. We demonstrated that the Bo homeolog of BnPOX1 was up-regulated after infection, while both BnPOX2 homeologs were down-regulated. A bias toward reciprocal expression of the homeologs of BnPOX1 in different organs in the natural allotetraploid of B. napus was also observed. These results suggest that subfunctionalization of the duplicated BnPOX genes after B. napus polyploidization as well as subneofunctionalization of the homeologs in response to this specific biotic stress has occurred. Retention of expression patterns in the diploid progenitors and the natural allotetraploid in some organs indicates that the function of peroxidase genes has been conserved during evolution.     

Requirement for the induced expression of a cell wall associated receptor kinase for survival during the pathogen response

Pathogen infection of angiosperms must rely on some interaction between the extracellular matrix (ECM) and the invading agent, and may be accompanied by signaling between the ECM and cytoplasm. An Arabidopsis cell wall associated receptor kinase (Wak1) has an amino-terminal domain that is tightly associated with the ECM, spans the plasma membrane and has a cytoplasmic protein kinase domain. Wak1 expression is induced when Arabidopsis plants are infected with pathogen, or when the pathogen response is stimulated either by exogenous salicylate (SA) or its analog 2,2-dichloroisonicotinic acid (INA). This Wak1 induction requires the positive regulator NPR1/NIM1. Thus Wak1 is a pathogen-related (PR) protein. Expression of an antisense and a dominant negative allele of Wak1 shows that induced expression of Wak1 is needed for a plant to survive if stimulated by INA. Ectopic expression of the entire Wak1, or the kinase domain alone, can provide resistance to otherwise lethal SA levels. These experiments suggest that Wak1 expression and other PR proteins are protecting plants from detrimental effects incurred during the pathogen response. These results provide a direct link between a protein kinase that could mediate signals from the ECM, to the events that are precipitated by a pathogen infection.     

Protection of canola (Brassica napus) against fungal pathogens by strains of biocontrol rhizobacteria

Strains of Pseudomonas fluorescens and Bacillus amyloliquefaciens, isolated from soybean rhizosphere, inhibited mycelial growth of Botrytis cinerea and Sclerotinia sclerotiorum, in vitro. Leaves from Brassica napus seedlings, pre-inoculated with either of these bacteria, exhibited systemic protection against fungal pathogens.     

Identification and functionality prediction of pathogenesis‐related protein 1 from legume family

The production and accumulation of pathogenesis‐related (PR) proteins in plants is one of the important responses to biotic and abiotic stress. Large number of identified PR proteins has been categorized into 17 functional families based on their structure, phylogenetics, and biological activities. However, they are not widely studied in legume crops. Using 29 PR1 proteins from Arabidopsis thaliana, as query, here we have predicted 92 candidate PR1 proteins through the PSI‐BLAST and HMMER programs. These candidate proteins were comprehensively analyzed with, multiple sequence alignment, domain architecture studies, signal peptide, and motif extraction followed by phylogenetic analysis. Further, response of two candidate PR1 proteins from chickpea against Fusarium oxysporum f.sp.ciceri attack was validated using qRT‐PCR followed by their 3D structure prediction. To decipher mode of action for PR1s, docking of pathogen extracellular matrix components along with fungal elicitors was performed with two chickpea PR1 proteins. Based on these findings, we propose carbohydrate to be the unique pathogen‐recognition feature for PR1 proteins and β‐glucanase activity via β‐glucan binding or modification.     

Features of the extracellular domain of the S-locus receptor kinase from Brassica

An S-receptor kinase (SRK) gene associated with self-incompatibility in a Brassica napus subsp. oleifera line has been characterized. The SRK-A14 cDNA shows the highest levels of homology in the 5 end to the SLG-A14 cDNA present at the same locus. RNA blot analysis shows that the SRK-A14 gene is expressed predominantly in the pistil, and at lower levels in the anthers. The predicted amino acid sequences from the extracellular domain of the SRK-A14 gene and three other SRK genes were compared. The different SRK extracellular domains were for the most part very similar, with the exception of two variable regions containing a high level of amino acid alterations. These extracellular domains also contain a region of similarity to the immunoglobulin domains present in members of the immunoglobulin superfamily. These findings may define regions of the SRK protein that are necessary for interactions between SRK and other proteins.     

Diversity and biocontrol potential of endophytic fungi in Brassica napus

This study was conducted to isolate endophytic fungi from oilseed rape (Brassica napus), to identify the fungal endophytes based on morphology and ITS (ITS1-5.8S rDNA-ITS2) sequences, and to evaluate their efficacy in suppression of the plant pathogenic fungi Sclerotinia sclerotiorum and Botrytis cinerea. Selected endophytic fungal isolates were further tested for promoting growth of oilseed rape in potting experiments. A total of 97 endophytic fungal isolates were obtained from roots (35), stems (49) and leaves (13) of B. napus. Forty fungal species were identified and most species (80%) belong to Ascomycota. The species composition is highly diversified with Simpson’s diversity index reaching 0.959. Alternaria alternata is the dominant species accounting for 12.4% of the isolates. Twenty-four isolates exhibited antifungal activity against S. sclerotiorum in dual cultures on potato dextrose agar forming inhibition zones of 3–17 mm in width. The culture filtrates of Aspergillus flavipes CanS-34A, Chaetomium globosum CanS-73, Clonostachys rosea CanS-43 and Leptosphaeria biglobosa CanS-51 in potato dextrose broth exhibited consistent and effective suppression of oilseed rape leaf blight caused by S. sclerotiorum. Fusarium oxysporum CanR-46 was detected capable of production of volatile organic compounds highly inhibitory to S. sclerotiorum and B. cinerea. Moreover, A. alternata CanL-18, Fusarium tricinctum CanR-70 and CanR-71r, and L. biglobosa CanS-51 exhibited growth-promoting effects on oilseed rape. These results suggest that B. napus harbors diversified endophytic fungi, from which potential biocontrol agents against S. sclerotiorum and B. cinerea, and for promoting growth of B. napus can be screened.     

The infection processes of Sclerotinia sclerotiorum in cotyledon tissue of a resistant and a susceptible genotype of Brassica napus

Background and Aims

Sclerotinia sclerotiorum can attack >400 plant species worldwide. Very few studies have investigated host–pathogen interactions at the plant surface and cellular level in resistant genotypes of oilseed rape/canola (Brassica napus).

Methods

Infection processes of S. sclerotiorum were examined on two B. napus genotypes, one resistant cultivar ‘Charlton’ and one susceptible ‘RQ001-02M2’ by light and scanning electron microscopy from 2 h to 8 d post-inoculation (dpi).

Key Results

The resistant ‘Charlton’ impeded fungal growth at 1, 2 and 3 dpi, suppressed formation of appresoria and infection cushions, caused extrusion of protoplast from hyphal cells and produced a hypersensitive reaction. At 8 dpi, whilst in ‘Charlton’ pathogen invasion was mainly confined to the upper epidermis, in the susceptible ‘RQ001-02M2’, colonization up to the spongy mesophyll cells was evident. Calcium oxalate crystals were found in the upper epidermis and in palisade cells in susceptible ‘RQ001-02M2’ at 6 dpi, and throughout leaf tissues at 8 dpi. In resistant ‘Charlton’, crystals were not observed at 6 dpi, whereas at 8 dpi they were mainly confined to the upper epidermis. Starch deposits were also more prevalent in ‘RQ001-02M2’.

Conclusions

This study demonstrates for the first time at the cellular level that resistance to S. sclerotiorum in B. napus is a result of retardation of pathogen development, both on the plant surface and within host tissues. The resistance mechanisms identified in this study will be useful for engineering disease-resistant genotypes and for developing markers for screening for resistance against this pathogen.     

Role of camalexin, indole glucosinolates, and side chain modification of glucosinolate-derived isothiocyanates in defense of Arabidopsis against Sclerotinia sclerotiorum

Plant secondary metabolites are known to facilitate interactions with a variety of beneficial and detrimental organisms, yet the contribution of specific metabolites to interactions with fungal pathogens is poorly understood. Here we show that, with respect to aliphatic glucosinolate‐derived isothiocyanates, toxicity against the pathogenic ascomycete Sclerotinia sclerotiorum depends on side chain structure. Genes associated with the formation of the secondary metabolites camalexin and glucosinolate were induced in Arabidopsis thaliana leaves challenged with the necrotrophic pathogen S. sclerotiorum. Unlike S. sclerotiorum, the closely related ascomycete Botrytis cinerea was not identified to induce genes associated with aliphatic glucosinolate biosynthesis in pathogen‐challenged leaves. Mutant plant lines deficient in camalexin, indole, or aliphatic glucosinolate biosynthesis were hypersusceptible to S. sclerotiorum, among them the myb28 mutant, which has a regulatory defect resulting in decreased production of long‐chained aliphatic glucosinolates. The antimicrobial activity of aliphatic glucosinolate‐derived isothiocyanates was dependent on side chain elongation and modification, with 8‐methylsulfinyloctyl isothiocyanate being most toxic to S. sclerotiorum. This information is important for microbial associations with cruciferous host plants and for metabolic engineering of pathogen defenses in cruciferous plants that produce short‐chained aliphatic glucosinolates.