Abstract
Cross-kingdom small RNA (sRNA) silencing has recently emerged as a mechanism facilitating fungal colonization and disease development. Here we characterized RNAi pathways in Zymoseptoria tritici, a major fungal pathogen of wheat, and assessed their contribution to pathogenesis. Computational analysis of fungal sRNA and host mRNA sequencing datasets was used to define the global sRNA populations in Z. tritici and predict their mRNA targets in wheat. 389 in planta-induced sRNA loci were identified. sRNAs generated from some of these loci were predicted to target wheat mRNAs including those potentially involved in pathogen defense. However, molecular approaches failed to validate targeting of selected wheat mRNAs by fungal sRNAs. Mutant strains of Z. tritici carrying deletions of genes encoding key components of RNAi such as Dicer-like (DCL) and Argonaute (AGO) proteins were generated, and virulence bioassays suggested that these are dispensable for full infection of wheat. Nonetheless, our results did suggest the existence of non-canonical DCL-independent pathway(s) for sRNA biogenesis in Z. tritici. dsRNA targeting essential fungal genes applied in vitro or generated from an RNA virus vector in planta in a procedure known as HIGS (Host-Induced Gene Silencing) was ineffective in preventing Z. tritici growth or disease. We also demonstrated that Z. tritici is incapable of dsRNA uptake. Collectively, our data suggest that RNAi approaches for gene function analyses in this fungal species and potentially also as a control measure may not be as effective as has been demonstrated for some other plant pathogenic fungi.
Highlights
In plants, RNA silencing known as RNAi (RNA interference) describes a collection of related biochemical pathways where numerous small RNA species, typically 21–24 nucleotides in length, e.g., miRNA, small interfering RNAs (siRNAs), tasiRNA, natsiRNA modify the expression of target mRNA molecules throughRNAi in Zymoseptoria tritici – Wheat Interactions classical Watson-Crick base-pairing (Ghildiyal and Zamore, 2009; Vickers et al, 2015)
Two candidate RNA-dependent RNA polymerase (RdRp) encoding genes, Mycgr3G51407 and Mycgr3G49833, were identified. All these genes encoding components of the RNAi machinery identified in the reference isolate IPO323 are present in the four Swiss Z. tritici isolates, 3D1, 1A5, 1E4, and 3D7, for which the genome data is available from Ensembl Fungi
Here we examined the possible role of cross-kingdom small RNA (sRNA) transfer and gene silencing in the interaction between this fungus and its natural host and assessed the potential of RNAi as a possible future strategy for control of septoria tritici blotch disease in wheat
Summary
RNA silencing known as RNAi (RNA interference) describes a collection of related biochemical pathways where numerous small RNA (sRNA) species, typically 21–24 nucleotides (nt) in length, e.g., miRNA (microRNA), siRNA (small interfering RNA), tasiRNA (trans-acting siRNA), natsiRNA (natural antisense siRNA) modify the expression of target mRNA molecules throughRNAi in Zymoseptoria tritici – Wheat Interactions classical Watson-Crick base-pairing (Ghildiyal and Zamore, 2009; Vickers et al, 2015). RNAi pathways are deeply integrated in plant immune processes and are involved in defense responses against viral, bacterial and fungal pathogens, and invertebrate pests (Hamilton and Baulcombe, 1999; Navarro et al, 2006; Pandey and Baldwin, 2007; Ellendorff et al, 2009; Kettles et al, 2013). They orchestrate numerous developmental processes, responses to changes in the abiotic environment, and DNA methylation and heterochromatin formation (Fujii et al, 2005; Matzke et al, 2007; Sunkar et al, 2007). RNAi pathways have been significantly modified or lost in some fungal lineages suggesting that in some circumstances loss of RNAi may confer significant evolutionary advantage (Billmyre et al, 2013; Nicolás et al, 2013)
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