Summary: Fusarium head blight (FHB) and Fusarium seedling blight (FSB) of wheat, caused by Fusarium pathogens, are devastating diseases worldwide. We report the expression of RNA interference (RNAi) sequences derived from an essential Fusarium graminearum (Fg) virulence gene, chitin synthase (Chs) 3b, as a method to enhance resistance of wheat plants to fungal pathogens. Deletion of Chs3b was lethal to Fg; disruption of the other Chs gene family members generated knockout mutants with diverse impacts on Fg. Comparative expression analyses revealed that among the Chs gene family members, Chs3b had the highest expression levels during Fg colonization of wheat. Three hairpin RNAi constructs corresponding to the different regions of Chs3b were found to silence Chs3b in transgenic Fg strains. Co‐expression of these three RNAi constructs in two independent elite wheat cultivar transgenic lines conferred high levels of stable, consistent resistance (combined type I and II resistance) to both FHB and FSB throughout the T₃ to T₅ generations. Confocal microscopy revealed profoundly restricted mycelia in Fg‐infected transgenic wheat plants. Presence of the three specific short interfering RNAs in transgenic wheat plants was confirmed by Northern blotting, and these RNAs efficiently down‐regulated Chs3b in the colonizing Fusarium pathogens on wheat seedlings and spikes. Our results demonstrate that host‐induced gene silencing of an essential fungal chitin synthase gene is an effective strategy for enhancing resistance in crop plants under field test conditions.

Summary: Chitin is a structural and functional component of the fungal cell wall and also serves as a pathogen‐associated molecular pattern (PAMP) that triggers the innate immune responses of host plants. However, no or very little chitin is found in the fungus‐like oomycetes. In Phytophthora spp., the presence of chitin has not been demonstrated so far, although putative chitin synthase (CHS) genes, which encode the enzymes that synthesize chitin, are present in their genomes. Here, we revealed that chitin is present in the zoospores and released sporangia of Phytophthora , and this is most consistent with the transcriptional pattern of PcCHS in Phytophthora capsici and PsCHS1 in Phytophthora sojae . Disruption of the CHS genes indicated that PcCHS and PsCHS1 , but not PsCHS2 (which exhibited very weak transcription), have similar functions involved in mycelial growth, sporangial production, zoospore release and the pathogenesis of P. capsici and P. sojae . We also suggest that chitin in the zoospores of P. capsici can act as a PAMP that is recognized by the chitin receptors AtLYK5 or AtCERK1 of Arabidopsis . These results provide new insights into the biological significance of chitin and CHSs in Phytophthora and help with the identification of potential targets for disease control.

Background: Plant transcription factors (TFs) are key transcriptional regulators to manipulate the regulatory network of host immunity. However, the globally transcriptional reprogramming of plant TF families in response to pathogens, especially between the resistant and susceptible host plants, remains largely unknown. Results: Here, we performed time-series RNA-seq from a resistant pepper line CM334 and a susceptible pepper line EC01 upon challenged with Phytophthora capsici, and enrichment analysis indicated that WRKY family most significantly enriched in both CM334 and EC01. Interestingly, we found that nearly half of the WRKY family members were significantly up-regulated, whereas none of them were down-regulated in the two lines. These induced WRKY genes were greatly overlapped between CM334 and EC01. More strikingly, most of these induced WRKY genes were expressed in time-order patterns, and could be mainly divided into three subgroups: early response (3 h-up), mid response (24 h-up) and mid-late response (ML-up) genes. Moreover, it was found that the responses of these ML-up genes were several hours delayed in EC01. Furthermore, a total of 19 induced WRKY genes were selected for functional identification by virus-induced gene silencing. The result revealed that silencing of CaWRKY03–6, CaWRKY03–7, CaWRKY06–5 or CaWRKY10–4 significantly increase the susceptibility to P. capsici both in CM334 and EC01, indicating that they might contribute to pepper’s basal defense against P. capsici; while silencing of CaWRKY08–4 and CaWRKY01–10 significantly impaired the disease resistance in CM334 but not in EC01, suggesting that these two WRKY genes are prominent modulators specifically in the resistant pepper plants. Conclusions: These results considerably extend our understanding of WRKY gene family in pepper’s resistance against P. capsici and provide potential applications for genetic improvement against phytophthora blight.

Phytophthora capsici is a broad–host range oomycete pathogen that can cause severe phytophthora blight disease of pepper and hundreds of other plant species worldwide. Natural resistance against P. capsici is inadequate, and it is very difficult to control by most of existing chemical fungicides. Therefore, it is urgent to develop alternative strategies to control this pathogen. Recently, host-induced or spray-induced gene silencing of essential or virulent pathogen genes provided an effective strategy for disease controls. Here, we demonstrate that P. capsici can effectively take up small interfering RNAs (siRNAs) from the environment. According to RNA-seq and quantitative reverse transcription PCR analysis, we identified four P. capsici RXLR effector genes that are significantly up-regulated during the infection stage. Transient overexpression and promote-infection assays indicated that RXLR1 and RXLR4 could promote pathogen infection. Using a virus-induced gene silencing system in pepper plants, we found that in planta–expressing RNA interference (RNAi) constructs that target RXLR1 or RXLR4 could significantly reduce pathogen infection, while co-interfering RXLR1 and RXLR4 could confer a more enhanced resistance to P. capsici. We also found that exogenously applying siRNAs that target RXLR1 or RXLR4 could restrict growth of P. capsici on the pepper and Nicotiana benthamiana leaves; when targeting RXLR1 and RXLR4 simultaneously, the control effect was more remarkable. These data suggested that RNAi-based gene silencing of RXLR effectors has great potential for application in crop improvement against P. capsici and also provides an important basis for the development of RNA-based antioomycete agents. [ ] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .