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Modulation of fungal virulence through CRZ1 regulated F-BAR-dependent actin remodeling and endocytosis in chickpea infecting phytopathogen Ascochyta rabiei.

ABSTRACT: Polarized hyphal growth of filamentous pathogenic fungi is an essential event for host penetration and colonization. The long-range early endosomal trafficking during hyphal growth is crucial for nutrient uptake, sensing of host-specific cues, and regulation of effector production. Bin1/Amphiphysin/Rvs167 (BAR) domain-containing proteins mediate fundamental cellular processes, including membrane remodeling and endocytosis. Here, we identified a F-BAR domain protein (ArF-BAR) in the necrotrophic fungus Ascochyta rabiei and demonstrate its involvement in endosome-dependent fungal virulence on the host plant Cicer arietinum. We show that ArF-BAR regulates endocytosis at the hyphal tip, localizes to the early endosomes, and is involved in actin dynamics. Functional studies involving gene knockout and complementation experiments reveal that ArF-BAR is necessary for virulence. The loss-of-function of ArF-BAR gene results in delayed formation of apical septum in fungal cells near growing hyphal tip that is crucial for host penetration, and impaired secretion of a candidate effector having secretory signal peptide for translocation across the endoplasmic reticulum membrane. The mRNA transcripts of ArF-BAR were induced in response to oxidative stress and infection. We also show that ArF-BAR is able to tubulate synthetic liposomes, suggesting the functional role of F-BAR domain in membrane tubule formation in vivo. Further, our studies identified a stress-induced transcription factor, ArCRZ1 (Calcineurin-responsive zinc finger 1), as key transcriptional regulator of ArF-BAR expression. We propose a model in which ArCRZ1 functions upstream of ArF-BAR to regulate A. rabiei virulence through a mechanism that involves endocytosis, effector secretion, and actin cytoskeleton regulation.

SUBMITTER: Sinha M

PROVIDER: S-EPMC8158962 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Transcription factors (TFs) are the key players in gene expression and their study is highly significant for shedding light on the molecular mechanisms and evolutionary history of organisms. During host-pathogen interaction, extensive reprogramming of gene expression facilitated by TFs is likely to occur in both host and pathogen. To date, the knowledge about TF repertoire in filamentous fungi is in infancy. The necrotrophic fungus Ascochyta rabiei, that causes destructive Ascochyta blight (AB) disease of chickpea (Cicer arietinum), demands more comprehensive study for better understanding of Ascochyta-legume pathosystem. In the present study, we performed the genome-wide identification and analysis of TFs in A. rabiei. Taking advantage of A. rabiei genome sequence, we used a bioinformatic approach to predict the TF repertoire of A. rabiei. For identification and classification of A. rabiei TFs, we designed a comprehensive pipeline using a combination of BLAST and InterProScan software. A total of 381 A. rabiei TFs were predicted and divided into 32 fungal specific families of TFs. The gene structure, domain organization and phylogenetic analysis of abundant families of A. rabiei TFs were also carried out. Comparative study of A. rabiei TFs with that of other necrotrophic, biotrophic, hemibiotrophic, symbiotic, and saprotrophic fungi was performed. It suggested presence of both conserved as well as unique features among them. Moreover, cis-acting elements on promoter sequences of earlier predicted A. rabiei secretome were also identified. With the help of published A. rabiei transcriptome data, the differential expression of TF and secretory protein coding genes was analyzed. Furthermore, comprehensive expression analysis of few selected A. rabiei TFs using quantitative real-time polymerase chain reaction revealed variety of expression patterns during host colonization. These genes were expressed in at least one of the time points tested post infection. Overall, this study illustrates the first genome-wide identification and analysis of TF repertoire of A. rabiei. This work would provide the basis for further studies to dissect role of TFs in the molecular mechanisms during A. rabiei-chickpea interactions.

Project description:Chickpea (Cicer arietinum L.) is an important cool season food legume, however, its production is severely constrained by the foliar disease Ascochyta blight caused by the fungus Ascochyta rabiei (syn. Phoma rabiei). Several disease management options have been developed to control the pathogen, including breeding for host plant resistance. However, the pathogen population is evolving to produce more aggressive isolates. For host resistance to be effective, the plant must quickly recognize the pathogen and instigate initial defense mechanisms, optimally at the point of contact. Given that the most resistant host genotypes display rapid pathogen recognition and response, the approach taken was to assess the type, speed and pattern of recognition via Resistance Gene Analog (RGA) transcription among resistant and susceptible cultivated chickpea varieties. RGAs are key factors in the recognition of plant pathogens and the signaling of inducible defenses. In this study, a suite of RGA loci were chosen for further investigation from both published literature and from newly mined homologous sequences within the National Center for Biotechnology Information (NCBI) database. Following their validation in the chickpea genome, 10 target RGAs were selected for differential expression analysis in response to A. rabiei infection. This was performed in a set of four chickpea varieties including two resistant cultivars (ICC3996 and PBA Seamer), one moderately resistant cultivar (PBA HatTrick) and one susceptible cultivar (Kyabra). Gene expression at each RGA locus was assessed via qPCR at 2, 6, and 24 h after A. rabiei inoculation with a previously characterized highly aggressive isolate. As a result, all loci were differentially transcribed in response to pathogen infection in at least one genotype and at least one time point after inoculation. Among these, the differential expression of four RGAs was significant and consistently increased in the most resistant genotype ICC3996 immediately following inoculation, when spore germination began and ahead of penetration into the plant's epidermal tissues. Further in silico analyses indicated that the differentially transcribed RGAs function through ADP-binding within the pathogen recognition pathway. These represent clear targets for future functional validation and potential for selective resistance breeding for introgression into elite cultivars.

Project description:Using microarray technology and a set of chickpea (Cicer arietinum L.) unigenes, grasspea (Lathyrus sativus L.) ESTs and lentil (Lens culinaris Med.) resistance gene analogs, the ascochyta blight (Ascochyta rabiei (Pass.) L.) resistance response was studied in four chickpea genotypes, including resistant, moderately resistant, susceptible and wild relative (Cicer echinospermum L.) genotypes. The experimental system minimized environmental effects and was conducted in reference design, where samples from mock-inoculated controls acted as references against post-inoculation samples. Robust data quality was achieved through the use of three biological replicates (including a dye-swap), the inclusion of negative controls, and strict selection criteria for differentially expressed genes including a fold change cutoff determined by self-self hybridizations, Students t test and multiple testing correction (P<0.05). Microarray observations were also validated by quantitative RT-PCR. The time-course expression patterns of 756 microarray features resulted in differential expression of 97 genes in at least one genotype at one time-point. K-means clustering grouped the genes into clusters of similar observations for each genotype, and comparisons between A. rabiei-resistant and susceptible genotypes revealed potential gene 'signatures' predictive of effective A. rabiei resistance. These genes included several pathogenesis-related proteins, SNAKIN2 antimicrobial peptide, proline-rich protein, disease resistance response protein DRRG49-C, environmental stress-inducible protein, leucine-zipper protein, polymorphic antigen membrane protein, as well as several unknown proteins. The potential involvement of these genes and their pathways of induction are discussed. This study represents the first large-scale gene expression profiling in chickpea, and future work will focus on functional validation of the genes of interest. Keywords: time course disease state analysis

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Modulation of fungal virulence through CRZ1 regulated F-BAR-dependent actin remodeling and endocytosis in chickpea infecting phytopathogen Ascochyta rabiei.

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