Necrotrophic Fungus Alternaria Brassicicola Research Articles

Identification of CmbHLH Transcription Factor Family and Excavation of CmbHLHs Resistant to Necrotrophic Fungus Alternaria in Chrysanthemum.

Chrysanthemum morifolium Ramat. 'Huaihuang' is a traditional Chinese medicinal plant. However, a black spot disease caused by Alternaria sp., a typical necrotrophic fungus, has a serious damaging influence on the field growth, yield, and quality of the plant. 'Huaiju 2#' being bred from 'Huaihuang', shows resistance to Alternaria sp. bHLH transcription factor has been widely studied because of their functions in growth development, signal transduction, and abiotic stress. However, the function of bHLH in biotic stress has rarely been studied. To characterize the resistance genes, the CmbHLH family was surveyed in 'Huaiju 2#'. On the basis of the transcriptome database of 'Huaiju 2#' after Alternaria sp. inoculation, with the aid of the Chrysanthemum genome database, 71 CmbHLH genes were identified and divided into 17 subfamilies. Most (64.8%) of the CmbHLH proteins were rich in negatively charged amino acids. CmbHLH proteins are generally hydrophilic proteins with a high aliphatic amino acid content. Among the 71 CmbHLH proteins, five CmbHLHs were significantly upregulated by Alternaria sp. infection, and the expression of CmbHLH18 was the most significant. Furthermore, heterologous overexpression of CmbHLH18 could improve the resistance of Arabidopsis thaliana to necrotrophic fungus Alternaria brassicicola by enhancing callose deposition, preventing spores from entering leaves, reducing ROS accumulation, increasing the activities of antioxidant enzymes and defense enzymes, and promoting their gene expression levels. These results indicate that the five CmbHLHs, especially CmbHLH18, may be considered candidate genes for resistance to necrotrophic fungus. These findings not only increase our understanding of the role CmbHLHs play in biotic stress but also provide a basis by using CmbHLHs to breed a new variety of Chrysanthemum with high resistance to necrotrophic fungus.

Seed Transmission of Pathogens: Non-Canonical Immune Response in Arabidopsis Germinating Seeds Compared to Early Seedlings against the Necrotrophic Fungus Alternaria brassicicola.

The transmission of seed-borne pathogens by the germinating seed is responsible for major crop diseases. The immune responses of the seed facing biotic invaders are poorly documented so far. The Arabidopsis thaliana/Alternaria brassicicola patho-system was used to describe at the transcription level the responses of germinating seeds and young seedling stages to infection by the necrotrophic fungus. RNA-seq analyses of healthy versus inoculated seeds at 3 days after sowing (DAS), stage of radicle emergence, and at 6 and 10 DAS, two stages of seedling establishment, identified thousands of differentially expressed genes by Alternaria infection. Response to hypoxia, ethylene and indole pathways were found to be induced by Alternaria in the germinating seeds. However, surprisingly, the defense responses, namely the salicylic acid (SA) pathway, the response to reactive oxygen species (ROS), the endoplasmic reticulum-associated protein degradation (ERAD) and programmed cell death, were found to be strongly induced only during the latter post-germination stages. We propose that this non-canonical immune response in early germinating seeds compared to early seedling establishment was potentially due to the seed-to-seedling transition phase. Phenotypic analyses of about 14 mutants altered in the main defense pathways illustrated these specific defense responses. The unexpected germination deficiency and insensitivity to Alternaria in the glucosinolate deficient mutants allow hypothesis of a trade-off between seed germination, necrosis induction and Alternaria transmission to the seedling. The imbalance of the SA and jasmonic acid (JA) pathways to the detriment of the JA also illustrated a non-canonical immune response at the first stages of the seedling.

In silico analysis of RNA interference components and miRNAs-like RNAs in the seed-borne necrotrophic fungus Alternariabrassicicola

RNA interference is a mechanism of suppressing gene expression in plants, animals and fungi. This regulation mechanism involves three main enzymes, Dicers (Dcr), Argonautes (Ago) and RNA Dependent RNA Polymerases (Rdrp) allowing to produce smallRNAs. RNA interference and smallRNAs have a role in the plant–microorganisms interaction, either in a pathogenic or in a symbiotic relationships. Alternaria brassicicola is a pathogenic fungus of the Brassicaceae plants. During plant infection, it is able to transmit itself vertically and horizontally, giving advantages for new infection and dissemination. To investigate RNA interference and the presence of smallRNAs in A. brassicicola, an in silico analysis was achieved. Two DCR, 4 AGO and 3 RDRP genes were identified comforting the presence of smallRNAs in A. brassicicola. SmallRNA sequencing from wild-type strain and DCR deleted mutants allowed the identifcation of 17 miRNAs in A. brassicicola. The synthesis of these miRNAs is only weakly influenced by the inactivation of DCR genes suggesting the possible existence of an alternative Dicer-independent miRNA synthesis pathway. Target's prediction of A. brassicicola miRNAs identified genes in the fungus and in the plant model Arabidopsis thaliana. Some miRNAs were predicted to target A. thaliana genes involved in the methylation of histone and in the disease resistance.

The Effect of Photoperiod on Necrosis Development, Photosynthetic Efficiency and 'Green Islands' Formation in Brassica juncea Infected with Alternaria brassicicola.

The main goal of growing plants under various photoperiods is to optimize photosynthesis for using the effect of day length that often acts on plants in combination with biotic and/or abiotic stresses. In this study, Brassica juncea plants were grown under four different day-length regimes, namely., 8 h day/16 h night, 12 h day/12 h night, 16 h day/8 h night, and continuous light, and were infected with a necrotrophic fungus Alternaria brassicicola. The development of necroses on B. juncea leaves was strongly influenced by leaf position and day length. The largest necroses were formed on plants grown under a 16 h day/8 h night photoperiod at 72 h post-inoculation (hpi). The implemented day-length regimes had a great impact on leaf morphology in response to A. brassicicola infection. They also influenced the chlorophyll and carotenoid contents and photosynthesis efficiency. Both the 1st (the oldest) and 3rd infected leaves showed significantly higher minimal fluorescence (F0) compared to the control leaves. Significantly lower values of other investigated chlorophyll a fluorescence parameters, e.g., maximum quantum yield of photosystem II (Fv/Fm) and non-photochemical quenching (NPQ), were observed in both infected leaves compared to the control, especially at 72 hpi. The oldest infected leaf, of approximately 30% of the B. juncea plants, grown under long-day and continuous light conditions showed a ‘green island’ phenotype in the form of a green ring surrounding an area of necrosis at 48 hpi. This phenomenon was also reflected in changes in the chloroplast’s ultrastructure and accelerated senescence (yellowing) in the form of expanding chlorosis. Further research should investigate the mechanism and physiological aspects of ‘green islands’ formation in this pathosystem.

Metapopulation Structure Predicts Population Dynamics in the Cakile maritima-Alternaria brassicicola Host-Pathogen Interaction.

AbstractIn symbiotic interactions, spatiotemporal variation in the distribution or population dynamics of one species represents spatial and temporal heterogeneity of the landscape for the other. Such interdependent demographic dynamics result in situations where the relative importance of biotic and abiotic factors in determining ecological processes is complicated to decipher. Using a detailed survey of three metapopulations of the succulent plant Cakile maritima and the necrotrophic fungus Alternaria brassicicola located along the southeastern Australian coast, we developed a series of statistical analyses-namely, synchrony analysis, patch occupancy dynamics, and a spatially explicit metapopulation model-to understand how habitat quality, weather conditions, dispersal, and spatial structure determine metapopulation dynamics. Climatic conditions are important drivers, likely explaining the high synchrony among populations. Host availability, landscape features facilitating dispersal, and habitat conditions also impact the occurrence and spread of disease. Overall, we show that the collection of extensive data on host and pathogen population dynamics, in combination with spatially explicit epidemiological modeling, makes it possible to accurately predict disease dynamics-even when there is extreme variability in host population dynamics. Finally, we discuss the importance of genetic information for predicting demographic dynamics in this pathosystem.

RLP23 is required for Arabidopsis immunity against the grey mould pathogen Botrytis cinerea

Necrosis- and ethylene-inducing-like proteins (NLPs) are secreted by fungi, oomycetes and bacteria. Conserved nlp peptides derived from NLPs are recognized as pathogen-associated molecular patterns (PAMPs), leading to PAMP-triggered immune responses. RLP23 is the receptor of the nlp peptides in Arabidopsis thaliana; however, its actual contribution to plant immunity is unclear. Here, we report that RLP23 is required for Arabidopsis immunity against the necrotrophic fungal pathogen Botrytis cinerea. Arabidopsis rlp23 mutants exhibited enhanced susceptibility to B. cinerea compared with the wild-type plants. Notably, microscopic observation of the B. cinerea infection behaviour indicated the involvement of RLP23 in pre-invasive resistance to the pathogen. B. cinerea carried two NLP genes, BcNEP1 and BcNEP2; BcNEP1 was expressed preferentially before/during invasion into Arabidopsis, whereas BcNEP2 was expressed at the late phase of infection. Importantly, the nlp peptides derived from both BcNEP1 and BcNEP2 induced the production of reactive oxygen species in an RLP23-dependent manner. In contrast, another necrotrophic fungus Alternaria brassicicola did not express the NLP gene in the early infection phase and exhibited no enhanced virulence in the rlp23 mutants. Collectively, these results strongly suggest that RLP23 contributes to Arabidopsis pre-invasive resistance to B. cinerea via NLP recognition at the early infection phase.

Omics-Based Mechanistic Insight Into the Role of Bioengineered Nanoparticles for Biotic Stress Amelioration by Modulating Plant Metabolic Pathways.

Bioengineered silver nanoparticles can emerge as a facile approach to combat plant pathogen, reducing the use of pesticides in an eco-friendly manner. The plants’ response during tripartite interaction of plant, pathogen, and nanoparticles remains largely unknown. This study demonstrated the use of bioengineered silver nanoparticles in combating black spot disease caused by necrotrophic fungus Alternaria brassicicola in Arabidopsis thaliana via foliar spray. The particles reduced disease severity by 70–80% at 5 μg/ml without showing phytotoxicity. It elicited plant immunity by a significant reduction in reactive oxygen species (ROS), decreases in stress enzymes by 0.6–19.8-fold, and emergence of autophagy. Comparative plant proteomics revealed 599 proteins expressed during the interaction, where 117 differential proteins were identified. Among different categories, proteins involved in bioenergy and metabolism were most abundant (44%), followed by proteins involved in plant defense (20%). Metabolic profiling by gas chromatography–mass spectroscopy yielded 39 metabolite derivatives in non-polar fraction and 25 in the polar fraction of plant extracts. It was observed that proteins involved in protein biogenesis and early plant defense were overexpressed to produce abundant antimicrobial metabolites and minimize ROS production. Bioengineered silver nanoparticles performed dual functions to combat pathogen attack by killing plant pathogen and eliciting immunity by altering plant defense proteome and metabolome.

Role of membrane compartment occupied by Can1 (MCC) and eisosome subdomains in plant pathogenicity of the necrotrophic fungus Alternaria brassicicola

BackgroundMCC/eisosomes are membrane microdomains that have been proposed to participate in the plasma membrane function in particular by regulating the homeostasis of lipids, promoting the recruitment of specific proteins and acting as provider of membrane reservoirs.ResultsHere we showed that several potential MCC/eisosomal protein encoding genes in the necrotrophic fungus A. brassicicola were overexpressed when germinated spores were exposed to antimicrobial defence compounds, osmotic and hydric stresses, which are major constraints encountered by the fungus during the plant colonization process. Mutants deficient for key MCC/eisosome components did not exhibit any enhanced susceptibility to phytoalexins and to applied stress conditions compared to the reference strain, except for a slight hypersensitivity of the ∆∆abpil1a-abpil1b strain to 2 M sorbitol. Depending on the considered mutants, we showed that the leaf and silique colonization processes were impaired by comparison to the wild-type, and assumed that these defects in aggressiveness were probably caused by a reduced appressorium formation rate.ConclusionsThis is the first study on the role of MCC/eisosomes in the pathogenic process of a plant pathogenic fungus. A link between these membrane domains and the fungus ability to form functional penetration structures was shown, providing new potential directions for plant disease control strategies.

Response of two Arabidopsis ecotypes Columbia-0 and Dijon-G to necrotrophic and biotrophic pathogens

Arabidopsis thaliana L. ecotype Dijon-G (Di-G) showed a different symptom development during pathogenesis compared to ecotype Columbia-0 (Col-0). Previously, it has been shown that Di-G has a higher susceptibility to necrotrophic fungus Alternaria brassicicola than Col-0. In this study, Di-G showed enhanced disease susceptibility to necrotrophic fungi Botrytis cinerea, Sclerotinia sclerotiorum, and Sclerotium rolfsii known to secrete oxalic acid (OA) as a pathogenicity factor. Treatment with 50 and 100 mM OA resulted in a more leaf tissue collapse in Di-G than in Col-0. The OA also up-regulated expression of the salicylic acid (SA)-inducible pathogenesis-related gene 1 (PR1) and down-regulated expression of the jasmonic acid/ethylene-inducible defensin PDF1.2 gene in Di-G. By contrast, Di-G was resistant to hemibiotrophic fungus Colletotrichum higginsianum and biotrophic Turnip crinkle virus (TCV) infections. Application of 0.5 mM SA resulted in a higher accumulation of endogenous SA and in a preferential expression of SA-responsive genes in Di-G. Salicylic acid accelerated OA-triggered plant cell death and attenuated PDF1.2 expression in Di-G. These results suggest that the enhanced susceptibility of Di-G to necrotrophic pathogen infections might be mediated by attenuated JA-ethylene defence signalling and/or heightened SA-related defence signalling. Interaction of SA-signalling with OA secretion might be also involved in the enhanced susceptibility of Di-G.

Responses to Hydric Stress in the Seed-Borne Necrotrophic Fungus Alternaria brassicicola.

Alternaria brassicicola is a necrotrophic fungus causing black spot disease and is an economically important seed-borne pathogen of cultivated brassicas. Seed transmission is a crucial component of its parasitic cycle as it promotes long-term survival and dispersal. Recent studies, conducted with the Arabidopsis thaliana/A. brassicicola pathosystem, showed that the level of susceptibility of the fungus to water stress strongly influenced its seed transmission ability. In this study, we gained further insights into the mechanisms involved in the seed infection process by analyzing the transcriptomic and metabolomic responses of germinated spores of A. brassicicola exposed to water stress. Then, the repertoire of putative hydrophilins, a group of proteins that are assumed to be involved in cellular dehydration tolerance, was established in A. brassicicola based on the expression data and additional structural and biochemical criteria. Phenotyping of single deletion mutants deficient for fungal hydrophilin-like proteins showed that they were affected in their transmission to A. thaliana seeds, although their aggressiveness on host vegetative tissues remained intact.

Beneficial and Pathogenic Arabidopsis Root-Interacting Fungi Differently Affect Auxin Levels and Responsive Genes During Early Infection.

Auxin (indole-3-acetic acid, IAA) is an important phytohormone involved in root growth and development. Root-interacting beneficial and pathogenic fungi utilize auxin and its target genes to manipulate the performance of their hosts for their own needs. In order to follow and visualize auxin effects in fungi-colonized Arabidopsis roots, we used the dual auxin reporter construct DR5::EGFP-DR5v2::tdTomato and fluorescence microscopy as well as LC-MS-based phytohormone analyses. We demonstrate that the beneficial endophytic fungi Piriformospora indica and Mortierella hyalina produce and accumulate IAA in their mycelia, in contrast to the phytopathogenic biotrophic fungus Verticillium dahliae and the necrotrophic fungus Alternaria brassicicola. Within 3 h after exposure of Arabidopsis roots to the pathogens, the signals of the auxin-responsive reporter genes disappeared. When exposed to P. indica, significantly higher auxin levels and stimulated expression of auxin-responsive reporter genes were detected both in lateral root primordia and the root elongation zone within 1 day. Elevated auxin levels were also present in the M. hyalina/Arabidopsis root interaction, but no downstream effects on auxin-responsive reporter genes were observed. However, the jasmonate level was strongly increased in the colonized roots. We propose that the lack of stimulated root growth upon infection with M. hyalina is not caused by the absence of auxin, but an inhibitory effect mediated by high jasmonate content.

Contribution of cell wall peroxidase‐ and NADPH oxidase‐derived reactive oxygen species to Alternaria brassicicola‐induced oxidative burst in Arabidopsis

SummaryCell wall peroxidases and plasma membrane‐localized NADPH oxidases are considered to be the main sources of the apoplastic oxidative burst in plants attacked by microbial pathogens. In spite of this established doctrine, approaches attempting a comparative, side‐by‐side analysis of the functions of extracellular reactive oxygen species (ROS) generated by the two enzymatic sources are scarce. Previously, we have reported the role of Arabidopsis NADPH oxidase RBOHD (respiratory burst oxidase homologue D) in plants challenged with the necrotrophic fungus Alternaria brassicicola. Here, we present results on the activity of apoplastic class III peroxidases PRX33 (At3g49110) and PRX34 (At3g49120) investigated in the same Arabidopsis–Alternaria pathosystem. ROS generated by Arabidopsis peroxidases PRX33 and PRX34 increase the necrotic symptoms and colonization success of A. brassicicola. In addition, the knockdown of PRX33 and PRX34 transcript levels leads to a reduced number of host cells showing an extracellular burst of ROS after inoculation with A. brassicicola. Our results also reveal an age‐dependent transcript distribution of ROS‐producing peroxidase and NADPH oxidase enzymes, and some potential new components of the RBOHD, PRX33 and PRX34 signalling networks.

Herbivory-responsive calmodulin-like protein CML9 does not guide jasmonate-mediated defenses in Arabidopsis thaliana.

Calcium is an important second messenger in plants that is released into the cytosol early after recognition of various environmental stimuli. Decoding of such calcium signals by calcium sensors is the key for the plant to react appropriately to each stimulus. Several members of Calmodulin-like proteins (CMLs) act as calcium sensors and some are known to mediate both abiotic and biotic stress responses. Here, we study the role of the Arabidopsis thaliana CML9 in different stress responses. CML9 was reported earlier as defense regulator against Pseudomonas syringae. In contrast to salicylic acid-mediated defense against biotrophic pathogens such as P. syringae, defenses against herbivores and necrotrophic fungi are mediated by jasmonates. We demonstrate that CML9 is induced upon wounding and feeding of the insect herbivore Spodoptera littoralis. However, neither different CML9 loss-of-function mutant lines nor overexpression lines were impaired upon insect feeding. No difference in herbivore-induced phytohormone elevation was detected in cml9 lines. The defense against the spider mite Tetranychus urticae was also unaffected. In addition, cml9 mutant lines showed a wild type-like reaction to the necrotrophic fungus Alternaria brassicicola. Thus, our data suggest that CML9 might be a regulator involved only in the defense against biotrophic pathogens, independent of jasmonates. In addition, our data challenge the involvement of CML9 in plant drought stress response. Taken together, we suggest that CML9 is a specialized rather than a general regulator of stress responses in Arabidopsis.

Folic acid induces salicylic acid-dependent immunity in Arabidopsis and enhances susceptibility to Alternaria brassicicola.

Folates are essential for one-carbon transfer reactions in all organisms and contribute, for example, to de novo DNA synthesis. Here, we detected the folate precursors 7,8-dihydropteroate (DHP) and 4-amino-4-deoxychorismate (ADC) in extracts from Arabidopsis thaliana plants by Fourier transform ion cyclotron resonance-mass spectrometry. The accumulation of DHP, but not ADC, was induced after infection of plants with Pseudomonas syringae delivering the effector protein AvrRpm1. Application of folic acid or the DHP precursor 7,8-dihydroneopterin (DHN) enhanced resistance in Arabidopsis to P. syringae and elevated the transcript accumulation of the salicylic acid (SA) marker gene pathogenesis-related1 in both the treated and systemic untreated leaves. DHN- and folic acid-induced systemic resistance was dependent on SA biosynthesis and signalling. Similar to SA, folic acid application locally enhanced Arabidopsis susceptibility to the necrotrophic fungus Alternaria brassicicola. Together, the data associate the folic acid pathway with innate immunity in Arabidopsis, simultaneously activating local and systemic SA-dependent resistance to P. syringae and suppressing local resistance to A. brassicicola.

Transcriptional responses of the Bdtf1-deletion mutant to the phytoalexin brassinin in the necrotrophic fungus Alternaria brassicicola.

Brassica species produce the antifungal indolyl compounds brassinin and its derivatives, during microbial infection. The fungal pathogen Alternaria brassicicola detoxifies brassinin and possibly its derivatives. This ability is an important property for the successful infection of brassicaceous plants. Previously, we identified a transcription factor, Bdtf1, essential for the detoxification of brassinin and full virulence. To discover genes that encode putative brassinin-digesting enzymes, we compared gene expression profiles between a mutant strain of the transcription factor and wild-type A. brassicicola under two different experimental conditions. A total of 170 and 388 genes were expressed at higher levels in the mutants than the wild type during the infection of host plants and saprophytic growth in the presence of brassinin, respectively. In contrast, 93 and 560 genes were expressed, respectively, at lower levels in the mutant than the wild type under the two conditions. Fifteen of these genes were expressed at lower levels in the mutant than in the wild type under both conditions. These genes were assumed to be important for the detoxification of brassinin and included Bdtf1 and 10 putative enzymes. This list of genes provides a resource for the discovery of enzyme-coding genes important in the chemical modification of brassinin.

Arabidopsis PECTIN METHYLESTERASEs Contribute to Immunity against Pseudomonas syringae

Pectins, major components of dicot cell walls, are synthesized in a heavily methylesterified form in the Golgi and are partially deesterified by pectin methylesterases (PMEs) upon export to the cell wall. PME activity is important for the virulence of the necrotrophic fungal pathogen Botrytis cinerea. Here, the roles of Arabidopsis PMEs in pattern-triggered immunity and immune responses to the necrotrophic fungus Alternaria brassicicola and the bacterial hemibiotroph Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) were studied. Plant PME activity increased during pattern-triggered immunity and after inoculation with either pathogen. The increase of PME activity in response to pathogen treatment was concomitant with a decrease in pectin methylesterification. The pathogen-induced PME activity did not require salicylic acid or ethylene signaling, but was dependent on jasmonic acid signaling. In the case of induction by A. brassicicola, the ethylene response factor, but not the MYC2 branch of jasmonic acid signaling, contributed to induction of PME activity, whereas in the case of induction by Pma ES4326, both branches contributed. There are 66 PME genes in Arabidopsis, suggesting extensive genetic redundancy. Nevertheless, selected pme single, double, triple and quadruple mutants allowed significantly more growth of Pma ES4326 than wild-type plants, indicating a role of PMEs in resistance to this pathogen. No decreases in total PME activity were detected in these pme mutants, suggesting that the determinant of immunity is not total PME activity; rather, it is some specific effect of PMEs such as changes in the pattern of pectin methylesterification.

The Bdtf1 Gene in Alternaria brassicicola Is Important in Detoxifying Brassinin and Maintaining Virulence on Brassica Species

Brassinin is an antifungal compound induced in Brassica plants after microbial infection. Molecular evidence is incomplete, however, in supporting the importance of brassinin in plant resistance to pathogens. To test the importance of brassinin in plant defense, we studied the functions of the gene Bdtf1 in the necrotrophic fungus Alternaria brassicicola. Several strains of mutants of this gene were weakly virulent on Brassica species, causing lesions 70% smaller in diameter than the wild type on three Brassica species. These mutants, however, were as virulent as the wild type on Arabidopsis thaliana. They were similar to the wild type in spore germination, colony morphology, and mycelial growth in nutrient-rich media, both with and without stress-inducing chemicals. Unlike wild-type A. brassicicola, however, the mutants failed to germinate and their hyphal growth was arrested in the presence of 200 μM brassinin. When grown in a medium containing 100 μM brassinin, wild-type mycelium entirely converted the brassinin into a nontoxic derivative, of which the precise chemical nature was not established. Mutants of the Bdtf1 gene were unable to perform this conversion. Our results support the hypothesis that the ability of A. brassicicola to detoxify brassinin is necessary for successful infection of Brassica species.

Dehydrin-like Proteins in the Necrotrophic Fungus Alternaria brassicicola Have a Role in Plant Pathogenesis and Stress Response

In this study, the roles of fungal dehydrin-like proteins in pathogenicity and protection against environmental stresses were investigated in the necrotrophic seed-borne fungus Alternaria brassicicola. Three proteins (called AbDhn1, AbDhn2 and AbDhn3), harbouring the asparagine-proline-arginine (DPR) signature pattern and sharing the characteristic features of fungal dehydrin-like proteins, were identified in the A. brassicicola genome. The expression of these genes was induced in response to various stresses and found to be regulated by the AbHog1 mitogen-activated protein kinase (MAPK) pathway. A knock-out approach showed that dehydrin-like proteins have an impact mainly on oxidative stress tolerance and on conidial survival upon exposure to high and freezing temperatures. The subcellular localization revealed that AbDhn1 and AbDhn2 were associated with peroxisomes, which is consistent with a possible perturbation of protective mechanisms to counteract oxidative stress and maintain the redox balance in AbDhn mutants. Finally, we show that the double deletion mutant ΔΔabdhn1-abdhn2 was highly compromised in its pathogenicity. By comparison to the wild-type, this mutant exhibited lower aggressiveness on B. oleracea leaves and a reduced capacity to be transmitted to Arabidopsis seeds via siliques. The double mutant was also affected with respect to conidiation, another crucial step in the epidemiology of the disease.

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.

Role of mannitol metabolism in the pathogenicity of the necrotrophic fungus Alternaria brassicicola

In this study, the physiological functions of fungal mannitol metabolism in the pathogenicity and protection against environmental stresses were investigated in the necrotrophic fungus Alternaria brassicicola. Mannitol metabolism was examined during infection of Brassica oleracea leaves by sequential HPLC quantification of the major soluble carbohydrates and expression analysis of genes encoding two proteins of mannitol metabolism, i.e., a mannitol dehydrogenase (AbMdh), and a mannitol-1-phosphate dehydrogenase (AbMpd). Knockout mutants deficient for AbMdh or AbMpd and a double mutant lacking both enzyme activities were constructed. Their capacity to cope with various oxidative and drought stresses and their pathogenic behavior were evaluated. Metabolic and gene expression profiling indicated an increase in mannitol production during plant infection. Depending on the mutants, distinct pathogenic processes, such as leaf and silique colonization, sporulation, survival on seeds, were impaired by comparison to the wild-type. This pathogenic alteration could be partly explained by the differential susceptibilities of mutants to oxidative and drought stresses. These results highlight the importance of mannitol metabolism with respect to the ability of A. brassicicola to efficiently accomplish key steps of its pathogenic life cycle.