Sharp eyespot, caused by necrotrophic fungus Rhizoctonia cerealis, is a serious fungal disease in wheat (Triticum aestivum). Certain wall-associated receptor kinases (WAK) mediate resistance to diseases caused by biotrophic/hemibiotrophic pathogens in several plant species. Yet, none of wheat WAK genes with positive effect on the innate immune responses to R. cerealis has been reported. In this study, we identified a WAK gene TaWAK7D, located on chromosome 7D, and showed its positive regulatory role in the defense response to R. cerealis infection in wheat. RNA-seq and qRT-PCR analyses showed that TaWAK7D transcript abundance was elevated in wheat after R. cerealis inoculation and the induction in the stem was the highest among the tested organs. Additionally, TaWAK7D transcript levels were significantly elevated by pectin and chitin treatments. The knock-down of TaWAK7D transcript impaired resistance to R. cerealis and repressed the expression of five pathogenesis-related genes in wheat. The green fluorescent protein signal distribution assays indicated that TaWAK7D localized on the plasma membrane in wheat protoplasts. Thus, TaWAK7D, which is induced by R. cerealis, pectin and chitin stimuli, positively participates in defense responses to R. cerealis through modulating the expression of several pathogenesis-related genes in wheat.

The domain of unknown function 26 (DUF26), harboring a conserved cysteine-rich motif (C-X8-C-X2-C), is unique to land plants. Several cysteine-rich repeat proteins (CRRs), belonging to DUF26-containing proteins, have been implicated in the defense against fungal pathogens in ginkgo, cotton, and maize. However, little is known about the functional roles of CRRs in the important staple crop wheat (Triticum aestivum). In this study, we identified a wheat CRR-encoding gene TaCRR1 through transcriptomic analysis, and dissected the defense role of TaCRR1 against the soil-borne fungi Rhizoctonia cerealis and Bipolaris sorokiniana, causal pathogens of destructive wheat diseases. TaCRR1 transcription was up-regulated in wheat towards B. Sorokiniana or R. cerealis infection. The deduced TaCRR1 protein contained a signal peptide and two DUF26 domains. Heterologously-expressed TaCRR1 protein markedly inhibited the mycelia growth of B. sorokiniana and R. cerealis. Furthermore, the silencing of TaCRR1 both impaired host resistance to B. sorokiniana and R. cerealis and repressed the expression of several pathogenesis-related genes in wheat. These results suggest that the TaCRR1 positively participated in wheat defense against both B. sorokiniana and R. cerealis through its antifungal activity and modulating expression of pathogenesis-related genes. Thus, TaCRR1 is a candidate gene for improving wheat resistance to B. sorokiniana and R. cerealis.

Fusarium head blight (FHB) and sharp eyespot are important diseases of the cereal plants, including bread wheat (Triticum aestivum) and barley. Both diseases are predominately caused by the pathogenic fungi, Fusarium graminearum and Rhizoctonia cerealis. The roles of the wheat-wall-associated kinases (WAKs) in defense against both F. graminearum and R. cerealis have remained largely unknown. This research reports the identification of TaWAK2A-800, a wheat WAK-coding gene located on chromosome 2A, and its functional roles in wheat resistance responses to FHB and sharp eyespot. TaWAK2A-800 transcript abundance was elevated by the early infection of R. cerealis and F. graminearum, or treatment with exogenous chitin. The gene transcript seemed to correspond to the resistance of wheat. Further functional analyses showed that silencing TaWAK2A-800 compromised the resistance of wheat to both FHB (F. graminearum) and sharp eyespot (R. cerealis). Moreover, the silencing reduced the expression levels of six defense-related genes, including the chitin-triggering immune pathway-marker genes, TaCERK1, TaRLCK1B, and TaMPK3. Summarily, TaWAK2A-800 participates positively in the resistance responses to both F. graminearum and R. cerealis, possibly through a chitin-induced pathway in wheat. TaWAK2A-800 will be useful for breeding wheat varieties with resistance to both FHB and sharp eyespot.

The fungus F. pseudograminearum can cause the destructive disease Fusarium crown rot (FCR) of wheat, an important staple crop. Functional roles of FCR resistance genes in wheat are largely unknown. In the current research, we characterized the antifungal activity and positive-regulatory function of the cysteine-rich repeat receptor-like kinase TaCRK-7A in the defense against F. pseudograminearum in wheat. Antifungal assays showed that the purified TaCRK-7A protein inhibited the growth of F. pseudograminearum. TaCRK-7A transcript abundance was elevated after F. pseudograminearum attack and was positively related to FCR-resistance levels of wheat cultivars. Intriguingly, knocking down of TaCRK-7A transcript increased susceptibility of wheat to FCR and decreased transcript levels of defense-marker genes in wheat. Furthermore, the transcript abundances of TaCRK-7A and its modulated-defense genes were responsive to exogenous jasmonate treatment. Taken together, these results suggest that TaCRK-7A can directly inhibit F. pseudograminearum growth and mediates FCR-resistance by promoting the expression of wheat defense genes in the jasmonate pathway. Thus, TaCRK-7A is a potential gene resource in FCR-resistant wheat breeding program.

Rhizoctonia cerealis is the causal agent of sharp eyespot, a devastating disease of cereal crops including wheat. Several metalloproteases have been implicated in pathogenic virulence, but little is known about whole-genome metalloproteases in R. cerealis. In this study, a total of 116 metalloproteases-encoding genes were identified and characterized from the R. cerealis Rc207 genome. The gene expression profiles and phylogenetic relationship of 11 MEP36/fungalysin metalloproteases were examined during the fungal infection to wheat, and function of an upregulated secretory MEP36 named RcFL1 was validated. Of 11 MEP36 family metalloproteases, ten, except RcFL5, were predicted to be secreted proteins and nine encoding genes, but not RcFL5 and RcFL2, were expressed during the R. cerealis infection process. Phylogenetic analysis suggested that MEP36 metalloproteases in R. cerealis were closely related to those of Rhizoctonia solani but were remote to those of Bipolaris sorokiniana, Fusarium graminearum, F. pseudograminearum, and Pyricularia oryzae. Expression of RcFL1 was significantly upregulated during the infection process and induced plant cell death in wheat to promote the virulence of the pathogen. The MEP36 domain was necessary for the activities of RcFL1. Furthermore, RcFL1 could repress the expression of wheat genes coding for the chitin elicitor receptor kinase TaCERK1 and chitinases. These results suggest that this MEP36 metalloprotease RcFL1 may function as a virulence factor of R. cerealis through inhibiting host chitin-triggered immunity and chitinases. This study provides insights on pathogenic mechanisms of R. cerealis. RcFL1 likely is an important gene resource for improving resistance of wheat to R. cerealis through host-induced gene silencing strategy.