TaRLK-6A promotes Fusarium crown rot resistance in wheat.

The plasma membrane-localized phytosulfokine receptor-like protein TaRLK-6A, interacting with TaSERK1, positively regulates the expression of defense-related genes in wheat, consequently promotes host resistance to Fusarium crown rot.

The Cytosolic Acetoacetyl-CoA Thiolase TaAACT1 Is Required for Defense against Fusarium pseudograminearum in Wheat.

Fusarium pseudograminearum is a major pathogen for the destructive disease Fusarium crown rot (FCR) of wheat (Triticum aestivum). The cytosolic Acetoacetyl-CoA thiolase II (AACT) is the first catalytic enzyme in the mevalonate pathway that biosynthesizes isoprenoids in plants. However, there has been no investigation of wheat cytosolic AACT genes in defense against pathogens including Fusarium pseudograminearum. Herein, we identified a cytosolic AACT-encoding gene from wheat, named TaAACT1, and demonstrated its positively regulatory role in the wheat defense response to F. pseudograminearum. One haplotype of TaAACT1 in analyzed wheat genotypes was associated with wheat resistance to FCR. The TaAACT1 transcript level was elevated after F. pseudograminearum infection, and was higher in FCR-resistant wheat genotypes than in susceptible wheat genotypes. Functional analysis indicated that knock down of TaAACT1 impaired resistance against F. pseudograminearum and reduced the expression of downstream defense genes in wheat. TaAACT1 protein was verified to localize in the cytosol of wheat cells. TaAACT1 and its modulated defense genes were rapidly responsive to exogenous jasmonate treatment. Collectively, TaAACT1 contributes to resistance to F. pseudograminearum through upregulating the expression of defense genes in wheat. This study sheds new light on the molecular mechanisms underlying wheat defense against FCR.

A Novel Wall-Associated Kinase TaWAK-5D600 Positively Participates in Defense against Sharp Eyespot and Fusarium Crown Rot in Wheat.

Sharp eyespot and Fusarium crown rot, mainly caused by soil-borne fungi Rhizoctonia cerealis and Fusarium pseudograminearum, are destructive diseases of major cereal crops including wheat (Triticum aestivum). However, the mechanisms underlying wheat-resistant responses to the two pathogens are largely elusive. In this study, we performed a genome-wide analysis of wall-associated kinase (WAK) family in wheat. As a result, a total of 140 TaWAK (not TaWAKL) candidate genes were identified from the wheat genome, each of which contains an N-terminal signal peptide, a galacturonan binding domain, an EGF-like domain, a calcium binding EGF domain (EGF-Ca), a transmembrane domain, and an intracellular Serine/Threonine protein kinase domain. By analyzing the RNA-sequencing data of wheat inoculated with R. cerealis and F. pseudograminearum, we found that transcript abundance of TaWAK-5D600 (TraesCS5D02G268600) on chromosome 5D was significantly upregulated, and that its upregulated transcript levels in response to both pathogens were higher compared with other TaWAK genes. Importantly, knock-down of TaWAK-5D600 transcript impaired wheat resistance against the fungal pathogens R. cerealis and F. pseudograminearum, and significantly repressed expression of defense-related genes in wheat, TaSERK1, TaMPK3, TaPR1, TaChitinase3, and TaChitinase4. Thus, this study proposes TaWAK-5D600 as a promising gene for improving wheat broad resistance to sharp eyespot and Fusarium crown rot (FCR) in wheat.

The GATA transcription factor TaGATA1 recruits demethylase TaELF6-A1 and enhances seed dormancy in wheat by directly regulating TaABI5.

Seed dormancy is an important agronomic trait in crops, and plants with low dormancy are prone to preharvest sprouting (PHS) under high-temperature and humid conditions. In this study, we report that the GATA transcription factor TaGATA1 is a positive regulator of seed dormancy by regulating TaABI5 expression in wheat. Our results demonstrate that TaGATA1 overexpression significantly enhances seed dormancy and increases resistance to PHS in wheat. Gene expression patterns, abscisic acid (ABA) response assay, and transcriptome analysis all indicate that TaGATA1 functions through the ABA signaling pathway. The transcript abundance of TaABI5, an essential regulator in the ABA signaling pathway, is significantly elevated in plants overexpressing TaGATA1. Chromatin immunoprecipitation assay (ChIP) and transient expression analysis showed that TaGATA1 binds to the GATA motifs at the promoter of TaABI5 and induces its expression. We also demonstrate that TaGATA1 physically interacts with the putative demethylase TaELF6-A1, the wheat orthologue of Arabidopsis ELF6. ChIP-qPCR analysis showed that H3K27me3 levels significantly decline at the TaABI5 promoter in the TaGATA1-overexpression wheat line and that transient expression of TaELF6-A1 reduces methylation levels at the TaABI5 promoter, increasing TaABI5 expression. These findings reveal a new transcription module, including TaGATA1-TaELF6-A1-TaABI5, which contributes to seed dormancy through the ABA signaling pathway and epigenetic reprogramming at the target site. TaGATA1 could be a candidate gene for improving PHS resistance.

The somatic embryogenesis receptor kinase TaSERK1 participates in the immune response to Rhizoctonia cerealis infection by interacting and phosphorylating the receptor-like cytoplasmic kinase TaRLCK1B in wheat.

The sharp eyespot, caused by necrotrophic pathogen Rhizoctonia cerealis, often causes serious yield loss in wheat (Triticum aestivum). However, the mechanisms underlying wheat resistant responses to the pathogen are still limited. In this study, we performed a genome-wide analysis of somatic embryogenesis receptor kinase (SERK) family in wheat. As a result, a total of 26 TaSERK candidate genes were identified from the wheat genome. Only 6 TaSERK genes on the chromosomes 2A, 2B, 2D, 3A, 3B, and 3D showed obvious heightening expression patterns in resistant wheat infected with R. cerealis compared than those un-infected wheat. Of them, the transcripts of 3 TaSERK1 homoeologs on the chromosomes 2A, 2B, and 2D were significantly up-regulated in the highest level compared to other TaSERKs. Importantly, silencing of TaSERK1 significantly impaired wheat resistance to sharp eyespot. Further bio-molecular assays showed that TaSERK1 could interact with the defence-associated receptor-like cytoplasmic kinase TaRLCK1B, and phosphorylated TaRLCK1B. Together, the results suggest that TaSERK1 mediated resistance responses to R. cerealis infection by interacting and phosphorylating TaRLCK1B in wheat. This study sheds light on the understanding of the wheat SERKs in the innate immunity against R. cerealis, and provided a theoretical fulcrum to identify candidate resistant genes for improving wheat resistance against sharp eyespot in wheat.

Whole-Genome Metalloproteases in the Wheat Sharp Eyespot Pathogen Rhizoctonia cerealis and a Role in Fungal Virulence.

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.

The L-Type Lectin-like Receptor Kinase Gene TaLecRK-IV.1 Regulates the Plant Height in Wheat.

Dwarfing is important for the production of wheat (Triticumaestivum L.). In model plants, receptor-like kinases have been implicated in signal transduction, immunity, and development. However, functional roles of lectin receptor-like kinases in wheat are poorly understood. In this study, we identified an L-type lectin receptor-like kinase gene in wheat, designated as TaLecRK-IV.1, and revealed its role in plant height. Real time quantitative PCR analyses indicated that TaLecRK-IV.1 transcript level was lower in a dwarf wheat line harboring the Rht-D1b gene compared to its transcript level detected in a taller wheat line CI12633. Importantly, the virus-induced gene silencing results showed that silencing of TaLecRK-IV.1 in the wheat line CI12633 led to dwarf plants. The results of the disease resistance test performed after the gene silencing experiment suggest no significant role of TaLecRK-IV.1 in the resistance reaction of wheat line CI12633 to sharp eyespot. Gene expression analysis revealed that the transcript abundance of TaLecRK-IV.1 was more up-regulated after the exogenous application of gibberellic acid and auxin, two development-related phytohormones, compared to the gene transcript levels detected in the control plants (mock treatment). These findings support the potential implication of TaLecRK-IV.1 in the pathway controlling plant height rather than the disease resistance role, and suggest that TaLecRK-IV.1 may be a positive regulator of plant height through the gibberellic acid and auxin-signaling pathways.

In situ analysis of acupuncture protecting dopaminergic neurons from lipid peroxidative damage in mice of Parkinson's disease.

OBJECTIVES: Acupuncture stimulation has proven to protect dopaminergic neurons from oxidative damage in animal models of Parkinson's disease (PD), but it remains unclear about the in situ information of biochemical components in dopaminergic neurons. Here, we aimed to analyse in situ changes of biochemical components and lipid peroxidation levels in dopaminergic neurons in PD mice treated with acupuncture by synchrotron FTIR micro-spectroscopy technique. MATERIALS AND METHODS: About 9-10-week-old C57BL/6 mice were used to establish PD model by intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 30 mg/kg for 5 days). Acupuncture stimulation was performed once a day for 12 days. Behaviour test was determined using the rotarod instrument. Biochemical compositions of dopaminergic neurons in substantia nigra pars compacta were analysed by synchrotron FTIR micro-spectroscopy technique. The number and ultrastructure of dopaminergic neurons were respectively observed by immunofluorescence and transmission electron microscopy (TEM). RESULTS: We found that the number and protein expression of dopaminergic neurons in MPTP-treated mice were reduced by about half, while that in the mice treated by acupuncture were significantly restored. Acupuncture treatment also restored the motor ability of PD mice. The results of single cell imaging with synchrotron FTIR micro-spectroscopy technique showed that the proportion of lipid in MPTP treated mice increased significantly. Especially the ratio of CH2 asymmetric stretching and CH3 asymmetric stretching increased significantly, suggesting that MPTP induced lipid peroxidation damage of dopaminergic neurons. It is also supported by the result of TEM, such as mitochondrial swelling or atrophy, loss of mitochondrial crests and mitochondrial vacuolization. Compared with MPTP treated mice, the proportion of lipid in acupuncture treated mice decreased and the mitochondrial structure was restored. CONCLUSIONS: Acupuncture can inhibit the level of lipid peroxides in dopaminergic neurons and protect neurons from oxidative damage. The study provides a promising method for in situ analysis of biochemical compositions in PD mice and reveals the mechanism of acupuncture in treating neurodegenerative diseases.

The Pathogen-Induced MATE Gene TaPIMA1 Is Required for Defense Responses to Rhizoctonia cerealis in Wheat.

The sharp eyespot, mainly caused by the soil-borne fungus Rhizoctonia cerealis, is a devastating disease endangering production of wheat (Triticum aestivum). Multi-Antimicrobial Extrusion (MATE) family genes are widely distributed in plant species, but little is known about MATE functions in wheat disease resistance. In this study, we identified TaPIMA1, a pathogen-induced MATE gene in wheat, from RNA-seq data. TaPIMA1 expression was induced by Rhizoctonia cerealis and was higher in sharp eyespot-resistant wheat genotypes than in susceptible wheat genotypes. Molecular biology assays showed that TaPIMA1 belonged to the MATE family, and the expressed protein could distribute in the cytoplasm and plasma membrane. Virus-Induced Gene Silencing plus disease assessment indicated that knock-down of TaPIMA1 impaired resistance of wheat to sharp eyespot and down-regulated the expression of defense genes (Defensin, PR10, PR1.2, and Chitinase3). Furthermore, TaPIMA1 was rapidly induced by exogenous H2O2 and jasmonate (JA) treatments, which also promoted the expression of pathogenesis-related genes. These results suggested that TaPIMA1 might positively regulate the defense against R. cerealis by up-regulating the expression of defense-associated genes in H2O2 and JA signal pathways. This study sheds light on the role of MATE transporter in wheat defense to Rhizoctonia cerealis and provides a potential gene for improving wheat resistance against sharp eyespot.

Overexpression of TaSTT3b-2B improves resistance to sharp eyespot and increases grain weight in wheat.

STAUROSPORINE AND TEMPERATURE SENSITIVE3 (STT3) is a catalytic subunit of oligosaccharyltransferase, which is important for asparagine-linked glycosylation. Sharp eyespot, caused by the necrotrophic fungal pathogen Rhizoctonia cerealis, is a devastating disease of bread wheat. However, the molecular mechanisms underlying wheat defense against R. cerealis are still largely unclear. In this study, we identified TaSTT3a and TaSTT3b, two STT3 subunit genes from wheat and reported their functional roles in wheat defense against R. cerealis and increasing grain weight. The transcript abundance of TaSTT3b-2B was associated with the degree of wheat resistance to R. cerealis and induced by both R. cerealis and exogenous jasmonic acid (JA). Overexpression of TaSTT3b-2B significantly enhanced resistance to R. cerealis, grain weight, and JA content in transgenic wheat subjected to R. cerealis stress, while silencing of TaSTT3b-2B compromised resistance of wheat to R. cerealis. Transcriptomic analysis showed that TaSTT3b-2B affected the expression of a series of defense-related genes and JA biosynthesis-related genes, as well as genes coding starch synthase and sucrose synthase. Application of exogenous JA elevated expression levels of the abovementioned defense- and grain weight-related genes, and rescuing the resistance of TaSTT3b-2B-silenced wheat to R. cerealis, while pretreatment with sodium diethyldithiocarbamate, an inhibitor of JA synthesis, attenuated the TaSTT3b-2B-mediated resistance to R. cerealis, suggesting that TaSTT3b-2B played critical roles in regulating R. cerealis resistance and grain weight via JA biosynthesis. Altogether, this study reveals new functional roles of TaSTT3b-2B in regulating plant innate immunity and grain weight, and illustrates its potential application value for wheat molecular breeding.

TaWAK2A-800, a Wall-Associated Kinase, Participates Positively in Resistance to Fusarium Head Blight and Sharp Eyespot in Wheat.

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 Receptor-like Kinase TaCRK-7A Inhibits Fusarium pseudograminearum Growth and Mediates Resistance to Fusarium Crown Rot in Wheat.

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.

The Wheat Wall-Associated Receptor-Like Kinase TaWAK-6D Mediates Broad Resistance to Two Fungal Pathogens Fusarium pseudograminearum and Rhizoctonia cerealis.

The soil-borne fungi Fusarium pseudograminearum and Rhizoctonia cerealis are the major pathogens for the economically important diseases Fusarium crown rot (FCR) and sharp eyespot of common wheat (Triticum aestivum), respectively. However, there has been no report on the broad resistance of wheat genes against both F. pseudograminearum and R. cerealis. In the current study, we identified TaWAK-6D, a wall-associated kinase (WAK) which is an encoding gene located on chromosome 6D, and demonstrated its broad resistance role in the wheat responses to both F. pseudograminearum and R. cerealis infection. TaWAK-6D transcript induction by F. pseudograminearum and R. cerealis was related to the resistance degree of wheat and the gene expression was significantly induced by exogenous pectin treatment. Silencing of TaWAK-6D compromised wheat resistance to F. pseudograminearum and R. cerealis, and repressed the expression of a serial of wheat defense-related genes. Ectopic expression of TaWAK-6D in Nicotiana benthamiana positively modulated the expression of several defense-related genes. TaWAK-6D protein was determined to localize to the plasma membrane in wheat and N. benthamiana. Collectively, the TaWAK-6D at the plasma membrane mediated the broad resistance responses to both F. pseudograminearum and R. cerealis in wheat at the seedling stage. This study, therefore, concludes that TaWAK-6D is a promising gene for improving wheat broad resistance to FCR and sharp eyespot.

The cysteine-rich receptor-like kinase TaCRK3 contributes to defense against Rhizoctonia cerealis in wheat.

Sharp eyespot, caused by the necrotrophic fungal pathogen Rhizoctonia cerealis, is a devastating disease of bread wheat (Triticum aestivum). However, the molecular mechanisms underlying wheat defense against R. cerealis are still largely unknown. In this study, by comparative transcriptomic analysis we identified a novel cysteine-rich receptor-like kinase (CRK)-encoding gene, designated as TaCRK3, and investigated its role in defense against R. cerealis. TaCRK3 transcript abundance was significantly elevated by R. cerealis and exogenous ethylene treatments. Silencing of TaCRK3 significantly compromised resistance to R. cerealis and repressed expression of an ethylene biosynthesis enzyme-encoding gene, ACO2, and a subset of defense-associated genes in wheat, whose transcript levels are up-regulated by ethylene stimulus. TaCRK3 protein was localized at the plasma membrane in wheat. Noticeably, both the heterologously expressed TaCRK3 protein and its partial peptide harboring two DUF26 (DOMAIN OF UNKNOWN FUNCTION 26) domains could inhibit growth of R. cerealis mycelia. These results suggest that TaCRK3 mediates wheat resistance to R. cerealis through direct antifungal activity and heightening the expression of defense-associated genes in the ethylene signaling pathway. Moreover, its DUF26 domains are required for the antifungal activity of TaCRK3. Our results reveal that TaCRK3 is a promising gene for breeding wheat varieties with resistance to R. cerealis.

The Wall-Associated Receptor-Like Kinase TaWAK7D Is Required for Defense Responses to Rhizoctonia cerealis in Wheat.

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.

Correction: Pan et al. 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. Int. J. Mol. Sci. 2020, 21, 2984.

In the original article, there was a mistake in Figure 8 as published [...].

The mitogen-activated protein kinase kinase TaMKK5 mediates immunity via the TaMKK5-TaMPK3-TaERF3 module.

Sharp eyespot disease, caused by the soil-borne fungus Rhizoctonia cerealis, seriously threatens production of wheat (Triticum aestivum). Despite considerable advances in understanding the mechanisms of mitogen-activated protein kinase (MAPK) cascades in innate immunity in model plant species, the roles of MAPK cascades in wheat are unknown. In this study, we identified a wheat MAPK kinase TaMKK5, located on chromosome 6B, and deciphered its functional role in the innate immune responses to R. cerealis attack. The TaMKK5-6B transcript level was elevated after R. cerealis infection and was higher in resistant wheat genotypes compared to susceptible genotypes. Overexpressing TaMKK5-6B increased resistance to sharp eyespot and upregulated the expression of multiple defense-related genes in wheat, including the MAPK gene TaMPK3, the ethylene response factor gene TaERF3, the calcium-dependent protein kinase gene TaCPK7-D, the glutathione s-transferase-1 gene TaGST1, Defensin, and Chitinase 2, while TaMKK5 knock-down compromised the resistance and repressed the expression of these defense-related genes. Bimolecular fluorescence complementation, yeast two-hybrid, pull-down, and phosphorylation assays showed that TaMKK5 physically interacted with TaMPK3, and phosphorylated and activated TaMPK3, and that TaMPK3 interacted with and phosphorylated TaERF3. The TaMKK5-TaMPK3 cascade modulates the expression of TaGST1, Defensin, and Chitinase 2 through TaERF3. Collectively, TaMKK5 mediates resistance to sharp eyespot through the TaMKK5-TaMPK3-TaERF3 module and by upregulating the expression of defense-related genes in wheat. This study provides insights into the role of the wheat MAPK cascades in innate immunity. TaMKK5-6B is a promising gene for breeding wheat cultivars that are resistant to sharp eyespot.

The Cysteine-Rich Repeat Protein TaCRR1 Participates in Defense against Both Rhizoctonia cerealis and Bipolaris sorokiniana 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.

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.

Rhizoctonia cerealis is the causal pathogen of the devastating disease, sharp eyespot, of the important crop wheat (Triticum aestivum L.). In phytopathogenic fungi, several M36 metalloproteases have been implicated in virulence, but pathogenesis roles of M35 family metalloproteases are largely unknown. Here, we identified four M35 family metalloproteases from R. cerealis genome, designated RcMEP2-RcMEP5, measured their transcriptional profiles, and investigated RcMEP2 function. RcMEP2-RcMEP5 are predicted as secreted metalloproteases since each protein sequence contains a signal peptide and an M35 domain that includes two characteristic motifs HEXXE and GTXDXXYG. Transcription levels of RcMEP2-RcMEP5 markedly elevated during the fungus infection to wheat, among which RcMEP2 expressed with the highest level. Functional dissection indicated that RcMEP2 and its M35 domain could trigger H2O2 rapidly-excessive accumulation, induce cell death, and inhibit expression of host chitinases. This consequently enhanced the susceptibility of wheat to R. cerealis and the predicated signal peptide of RcMEP2 functions required for secretion and cell death-induction. These results demonstrate that RcMEP2 is a virulence factor and that its M35 domain and signal peptide are necessary for the virulence role of RcMEP2. This study facilitates a better understanding of the pathogenesis mechanism of metalloproteases in phytopathogens including R. cerealis.

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

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.