Global Characterization of GH10 Family Xylanase Genes in Rhizoctonia cerealis and Functional Analysis of Xylanase RcXYN1 During Fungus Infection in Wheat

Abstract

Wheat (Triticum aestivum L.) is an important staple crop. Rhizoctonia cerealis is the causal agent of diseases that are devastating to cereal crops, including wheat. Xylanases play an important role in pathogenic infection, but little is known about xylanases in R. cerealis. Herein, we identified nine xylanase-encoding genes from the R. cerealis genome, named RcXYN1–RcXYN9, examined their expression patterns, and investigated the pathogenicity role of RcXYN1. RcXYN1–RcXYN9 proteins contain two conserved glutamate residues within the active motif in the glycoside hydrolase 10 (GH10) domain. Of them, RcXYN1–RcXYN4 are predicted to be secreted proteins. RcXYN1–RcXYN9 displayed different expression patterns during the infection process of wheat, and RcXYN1, RcXYN2, RcXYN5, and RcXYN9 were expressed highly across all the tested inoculation points. Functional dissection indicated that the RcXYN1 protein was able to induce necrosis/cell-death and H2O2 generation when infiltrated into wheat and Nicotiana benthamiana leaves. Furthermore, application of RcXYN1 protein followed by R. cerealis led to significantly higher levels of the disease in wheat leaves than application of the fungus alone. These results demonstrate that RcXYN1 acts as a pathogenicity factor during R. cerealis infection in wheat. This is the first investigation of xylanase genes in R. cerealis, providing novel insights into the pathogenesis mechanisms of R. cerealis.

Genome-Wide Identification of M35 Family Metalloproteases in Rhizoctonia cerealis and Functional Analysis of RcMEP2 as a Virulence Factor during the Fungal Infection to Wheat

“…In other phytopathogenic fungi, several M36 metalloproteases have been evidenced to be important virulence factors or effectors [27][28][29][30]32,33]. Our previous studies showed that the R. cerealis cutinase RcCUT1 and the R. cerealis xylanase RcXYN1 could trigger plant cell death and H 2 O 2 accumulation, and upregulated transcriptions of several defense-related genes in protein-treated wheat plants, leading to enhanced disease [11,12]. Here, sequence analysis showed RcMEP2 might be a secreted protein.…”

“…Using the purified RcMEP2 protein infiltrated into wheat and N. benthamiana leaves, our results reveal that the RcMEP2 is capable of inducing cell death and triggering H 2 O 2 rapidly-excessive accumulation in the treated leaves. Unlike the induction effect of the cutinase RcCUT1 and xylanase RcXYN1 of R. cerealis on transcriptions of several defense-related genes in these protein-treated wheat plants [11,12], RcMEP2 did not upregulate the transcriptions of these host defense-related genes and repressed the expression of wheat chitinases, implying distinct mechanisms underlying the roles of RcMEP2, RcCUT1, and RcXYN1. As expected, RcMEP2 treatment significantly increased disease severity compared to the negative controls, indicating that RcMEP2 treatment enhances virulence of R. cerealis during the fungal infection to wheat.…”

“…It was reported that the M36 metalloprotease-encoding gene Cgfl was strongly upregulated during early infection stages and acted as an effector to enhance virulence in the maize anthracnose fungus C. graminicola [27,28]. Additionally, the R. cerealis cutinase-encoding gene RcCUT1 and the R. cerealis xylanase-encoding gene RcXYN1 displayed significant upregulations during the fungus infection process to wheat [11,12]. Taken together, we deduced that RcMEP2 might be the most important one of the four M35 metalloproteases during the fungal infection to wheat, and further investigated the functional role of RcMEP2.…”

“…The recombinant His-TF-RcMEP2, His-TF-RcMEP2-1, and His-TF-RcMEP2-2 proteins were separately expressed after induction with 0.5 mM isopropyl-β-D-thiogalactopyranose at 16 • C for 12 h, and purified using Ni + resin (TransGen Biotech, Beijing, China). Protein purity and molecular weight were determined by using SDS-PAGE according to the method described by Lu et al [12].…”

“…Recently, we completed the genome sequencing of the R. cerealis strain Rc207 and finished genome de novo assembling and annotation, and completed time-course-infected transcriptome sequencing (Zengyan Zhang s lab, unpublished data), which establishes databases for further study. In addition, our recent papers showed that by means of the purified proteins infiltrated into leaves of wheat and tobacco, a cutinase RcCUT1 and a xylanase RcXYN1 functioned as effectors in R. cerealis infection [11,12]. However, the functional roles of several hundred candidate effectors of R. cerealis remain largely unknown.…”