Dataset Information

Cell-Wall-Degrading Enzymes Required for Virulence in the Host Selective Toxin-Producing Necrotroph Alternaria alternata of Citrus.

ABSTRACT: The necrotrophic fungal pathogen Alternaria alternata attacks many citrus species, causing brown spot disease. Its pathogenic capability depends primarily on the production of host-selective ACT toxin. In the current study a Ste12 transcription factor was characterized to be required for conidial formation and the production of cell-wall-degrading enzymes (CWDEs) in the tangerine pathotype of A. alternata. The Ste12 deficiency strain (?Ste12) retained wild-type growth, ACT toxin production, and sensitivity to oxidative and osmotic stress. However, pathogenicity tests assayed on detached Dancy leaves revealed a marked reduction in virulence of ?Ste12. Transcriptome and quantitative RT-PCR analyses revealed that many genes associated with Carbohydrate-Active Enzymes (CAZymes) were downregulated in ?Ste12. Two cutinase-coding genes (AaCut3 and AaCut7) regulated by Ste12 were individually and simultaneously inactivated. The AaCut3 or AaCut7 deficiency strain unchanged in cutinase activities and incited wild-type lesions on Dancy leaves. However, the strain carrying an AaCut3 AaCut7 double mutation produced and secreted significantly fewer cutinases and incited smaller necrotic lesions than wild type. Not only is the host-selective toxin (HST) produced by A. alternata required for fungal penetration and lesion formation, but so too are CWDEs required for full virulence. Overall, this study expands our understanding of how A. alternata overcomes citrus physical barriers to carry out successful penetration and colonization.

SUBMITTER: Ma H

PROVIDER: S-EPMC6883767 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Cell-Wall-Degrading Enzymes Required for Virulence in the Host Selective Toxin-Producing Necrotroph Alternaria alternata of Citrus.

Ma Haijie H Zhang Bin B Gai Yunpeng Y Sun Xuepeng X Chung Kuang-Ren KR Li Hongye H

Frontiers in microbiology 20191122

The necrotrophic fungal pathogen Alternaria alternata attacks many citrus species, causing brown spot disease. Its pathogenic capability depends primarily on the production of host-selective ACT toxin. In the current study a Ste12 transcription factor was characterized to be required for conidial formation and the production of cell-wall-degrading enzymes (CWDEs) in the tangerine pathotype of A. alternata. The Ste12 deficiency strain (ΔSte12) retained wild-type growth, ACT toxin pr ...[more]

PMID: 31824437

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Project description:The tangerine pathotype of Alternaria alternata produces a host-selective toxin termed Alternaria citri toxin (ACT). The molecular mechanisms underlying the global regulation and biosynthesis of ACT remain unknown. In the present study, the function of an APSES transcription factor StuA was investigated. StuA was shown to be required for ACT biosynthesis and fungal virulence. StuA was found, for the first time, to physically interact with a pH-responsive transcription regulator PacC using yeast two-hybrid, bimolecular fluorescence complementation, and GST pull-down assays. Functional analyses revealed that StuA and PacC regulate the expression of genes involved in toxin biosynthesis and virulence. Mutation of stuA via targeted gene deletion or silencing pacC yielded fungal strains that decreased the expression of seven toxin biosynthetic genes (ACCT) and toxin production. EMSA analyses revealed that PacC could bind to the promoters of ACTT6 encoding an enoyl-CoA hydratase and ACTTR encoding an ACT pathway-specific transcription factor. Site-directed mutagenesis of five potential protein kinase A (PKA) phosphorylation sites in StuA revealed that none of the sites was involved in ACT production, indicating that the function of StuA in the regulation of ACT gene expression is not dependent on phosphorylation. Overall, our results confirmed that PacC is one of the key regulators interacting with StuA for the biosynthesis of ACT. Environmental pH may play a decisive role during A. alternata pathogenesis. Our results also revealed a previously unrecognized (StuA-PacC)→ACTTR module for the biosynthesis of ACT in A. alternata. IMPORTANCE In this study, we used Alternaria alternata as a biological model to report the role of StuA in phytopathogenic fungi. Our findings indicated that StuA is required for Alternaria citri toxin (ACT) biosynthesis and fungal virulence. In addition, StuA physically interacts with PacC. Disruption of stuA or pacC led to decreased expression of seven toxin biosynthetic genes (ACCT) and toxin production. PacC could recognize and bind to the promoter regions of ACTT6 and ACTTR. Our results revealed a previously unrecognized (StuA-PacC)→ACTTR module for the biosynthesis of ACT in A. alternata, which also provides a framework for the study of StuA in other fungi.

Project description:Identifying a viable substitute for the limited array of current antifungal agents stands as a crucial objective in modern agriculture. Consequently, extensive worldwide research has been undertaken to unveil eco-friendly and effective agents capable of controlling pathogens resistant to the presently employed fungicides. This study explores the efficacy of Trichoderma isolates in combating tomato leaf spot disease, primarily caused by Alternaria alternata. The identified pathogen, A. alternata Alt3, was isolated and confirmed through the ITS region (OQ888806). Six Trichoderma isolates were assessed for their ability to inhibit Alt3 hyphal growth using dual culture, ethyl acetate extract, and volatile organic compounds (VOCs) techniques. The most promising biocontrol isolate was identified as T. afroharzianum isolate TRI07 based on three markers: ITS region (OQ820171), translation elongation factor alpha 1 gene (OR125580), and RNA polymerase II subunit gene (OR125581). The ethyl acetate extract of TRI07 isolate was subjected to GC-MS analysis, revealing spathulenol, triacetin, and aspartame as the main compounds, with percentages of 28.90, 14.03, and 12.97%, respectively. Analysis of TRI07-VOCs by solid-phase microextraction technique indicated that the most abundant compounds included ethanol, hydroperoxide, 1-methylhexyl, and 1-octen-3-one. When TRI07 interacted with Alt3, 34 compounds were identified, with major components including 1-octen-3-one, ethanol, and hexanedioic acid, bis(2-ethylhexyl) ester. In greenhouse experiment, the treatment of TRI07 48 h before inoculation with A. alternata (A3 treatment) resulted in a reduction in disease severity (16.66%) and incidence (44.44%). Furthermore, A3 treatment led to improved tomato growth performance parameters and increased chlorophyll content. After 21 days post-inoculation, A3 treatment was associated with increased production of antioxidant enzymes (CAT, POD, SOD, and PPO), while infected tomato plants exhibited elevated levels of oxidative stress markers MDA and H2O2. HPLC analysis of tomato leaf extracts from A3 treatment revealed higher levels of phenolic acids such as gallic, chlorogenic, caffeic, syringic, and coumaric acids, as well as flavonoid compounds including catechin, rutin, and vanillin. The novelty lies in bridging the gap between strain-specific attributes and practical application, enhancing the understanding of TRI07's potential for integrated pest management. This study concludes that TRI07 isolate presents potential natural compounds with biological activity, effectively controlling tomato leaf spot disease and promoting tomato plant growth. The findings have practical implications for agriculture, suggesting a sustainable biocontrol strategy that can enhance crop resilience and contribute to integrated pest management practices.

Project description:This study determined the function of thioredoxin and glutaredoxin systems in the phytopathogenic fungus Alternaria alternata via analyzing mutants obtained from the targeted deletion of genes encoding thioredoxin peroxidase (Tsa1), thioredoxin reductase (Trr1), and glutathione reductase (Glr1). Trr1 and Glr1, but not Tsa1, are required for growth and conidiation. The reduced growth and conidiation seen in the Trr1 or Glr1 deletion mutant can be restored by glutathione. Deletion mutants showing growth inhibition by oxidants are defective for H2O2 detoxification and induce smaller lesions on citrus leaves. Trr1 and Glr1, but not Tsa1, also contribute to NaCl resistance. Glr1 is required for sorbitol resistance and is responsible for resistance to mancozeb and boscalid but not chlorothalonil fungicides, a novel phenotype that has not been reported in fungi. Trr1 is required for resistance to boscalid and chlorothalonil fungicides but confers susceptibility to mancozeb. The Tsa1 deletion mutant displays wild-type sensitivity to the tested fungicides. The expression of Tsa1 and Trr1 is regulated by the oxidative stress responsive regulators Yap1, Hog1, and Skn7. The expression of Tsa1, but not Trr1, is also regulated indirectly by the NADPH oxidase. The results indicate that the capability to resist oxidative stress is required for virulence of A. alternataIMPORTANCE The thioredoxin and glutaredoxin systems are important thiol antioxidant systems in cells, and knowledge of these two systems in the plant-pathogenic fungus A. alternata is useful for finding new strategies to reduce the virulence of this pathogen. In this study, we demonstrated that thiol antioxidant system-related genes (Tsa1, Trr1, and Glr1) are required for H2O2 detoxification and virulence in A. alternata Moreover, deletion of Trr1 results in hypersensitivity to the fungicides chlorothalonil and boscalid, and Glr1 deletion mutants are highly sensitive to mancozeb, which is the fungicide mostly used in citrus fields. Therefore, our findings demonstrate that the ability to detoxify reactive oxygen species (ROS) plays a critical role in pathogenesis on citrus and provide novel insights into the physiological functions of thiol-containing systems in fungicide sensitivity for A. alternata.

Dataset Information

Cell-Wall-Degrading Enzymes Required for Virulence in the Host Selective Toxin-Producing Necrotroph Alternaria alternata of Citrus.

Publications

Cell-Wall-Degrading Enzymes Required for Virulence in the Host Selective Toxin-Producing Necrotroph <i>Alternaria alternata</i> of Citrus.

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