Identification of effector candidate genes of Rhizoctonia solani AG-1 IA expressed during infection in Brachypodium distachyon

Rhizoctonia solani is a necrotrophic phytopathogen belonging to basidiomycetes. It causes rice sheath blight which inflicts serious damage in rice production. The infection strategy of this pathogen remains unclear. We previously demonstrated that salicylic acid-induced immunity could block R. solani AG-1 IA infection in both rice and Brachypodium distachyon. R. solani may undergo biotrophic process using effector proteins to suppress host immunity before necrotrophic stage. To identify pathogen genes expressed at the early infection process, here we developed an inoculation method using B. distachyon which enables to sample an increased amount of semi-synchronous infection hyphae. Sixty-one R. solani secretory effector-like protein genes (RsSEPGs) were identified using in silico approach with the publicly available gene annotation of R. solani AG-1 IA genome and our RNA-sequencing results obtained from hyphae grown on agar medium. Expression of RsSEPGs was analyzed at 6, 10, 16, 24, and 32 h after inoculation by a quantitative reverse transcription-polymerase chain reaction and 52 genes could be detected at least on a single time point tested. Their expressions showed phase-specific patterns which were classified into 6 clusters. The 23 RsSEPGs in the cluster 1-3 and 29 RsSEPGs in the cluster 4-6 are expected to be involved in biotrophic and necrotrophic interactions, respectively

Time-series transcriptome of Brachypodium distachyon during bacterial flagellin-induced pattern-triggered immunity

Plants protect themselves from microorganisms by inducing pattern-triggered immunity (PTI) via recognizing microbe-associated molecular patterns (MAMPs), conserved across many microbes. Although the MAMP perception mechanism and initial events during PTI have been well-characterized, knowledge of the transcriptomic changes in plants, especially monocots, is limited during the intermediate and terminal stages of PTI. Here, we report a time-series high-resolution RNA-sequencing (RNA-seq) analysis during PTI in the leaf disks of Brachypodium distachyon. We identified 6,039 differentially expressed genes (DEGs) in leaves sampled at 0, 0.5, 1, 3, 6, and 12 hours after treatment (hat) with the bacterial flagellin peptide flg22. The k-means clustering method classified these DEGs into 10 clusters (6 upregulated and 4 downregulated). Based on the results, we selected 10 PTI marker genes in B. distachyon. Gene ontology (GO) analysis suggested a tradeoff between defense responses and photosynthesis during PTI. The data indicated the recovery of photosynthesis started at least at 12 hat. Over-representation analysis of transcription factor genes and cis-regulatory elements in DEG promoters implied the contribution of 12 WRKY transcription factors in plant defense at the early stage of PTI induction

BdWRKY38 is required for the incompatible interaction of Brachypodium distachyon with the necrotrophic fungus Rhizoctonia solani

Rhizoctonia solani is a soil‐borne necrotrophic fungus that causes sheath blight in grasses. The basal resistance of compatible interactions between R. solani and rice is known to be modulated by some WRKY transcription factors (TFs). However, genes and defense responses involved in incompatible interaction with R. solani remain unexplored, because no such interactions are known in any host plants. Recently, we demonstrated that Bd3‐1, an accession of the model grass Brachypodium distachyon, is resistant to R. solani and, upon inoculation with the fungus, undergoes rapid induction of genes responsive to the phytohormone salicylic acid (SA) that encode the WRKY TFs BdWRKY38 and BdWRKY44. Here, we show that endogenous SA and these WRKY TFs positively regulate this accession‐specific R. solani resistance. In contrast to a susceptible accession (Bd21), the infection process in the resistant accessions Bd3‐1 and Tek‐3 was suppressed at early stages before the development of fungal biomass and infection machinery. A comparative transcriptome analysis during pathogen infection revealed that putative WRKY‐dependent defense genes were induced faster in the resistant accessions than in Bd21. A gene regulatory network (GRN) analysis based on the transcriptome dataset demonstrated that BdWRKY38 was a GRN hub connected to many target genes specifically in resistant accessions, whereas BdWRKY44 was shared in the GRNs of all three accessions. Moreover, overexpression of BdWRKY38 increased R. solani resistance in Bd21. Our findings demonstrate that these resistant accessions can activate an incompatible host response to R. solani, and BdWRKY38 regulates this response by mediating SA signaling

Parental legacy and regulatory novelty in Brachypodium diurnal transcriptomes accompanying their polyploidy

Polyploidy is a widespread phenomenon in eukaryotes that can lead to phenotypic novelty and has important implications for evolution and diversification. The modification of phenotypes in polyploids relative to their diploid progenitors may be associated with altered gene expression. However, it is largely unknown how interactions between duplicated genes affect their diurnal expression in allopolyploid species. In this study, we explored parental legacy and hybrid novelty in the transcriptomes of an allopolyploid species and its diploid progenitors. We compared the diurnal transcriptomes of representative Brachypodium cytotypes, including the allotetraploid Brachypodium hybridum and its diploid progenitors Brachypodium distachyon and Brachypodium stacei. We also artificially induced an autotetraploid B. distachyon. We identified patterns of homoeolog expression bias (HEB) across Brachypodium cytotypes and time-dependent gain and loss of HEB in B. hybridum. Furthermore, we established that many genes with diurnal expression experienced HEB, while their expression patterns and peak times were correlated between homoeologs in B. hybridum relative to B. distachyon and B. stacei, suggesting diurnal synchronization of homoeolog expression in B. hybridum. Our findings provide insight into the parental legacy and hybrid novelty associated with polyploidy in Brachypodium, and highlight the evolutionary consequences of diurnal transcriptional regulation that accompanied allopolyploidy

A Seed-Borne Bacterium of Rice, Pantoea dispersa BB1, Protects Rice from the Seedling Rot Caused by the Bacterial Pathogen Burkholderia glumae

Seedling rot, caused by the bacterial pathogen Burkholderia glumae, is a major disease of rice. It originates from pathogen-contaminated seeds and is thus mainly controlled by pesticide treatments of seeds. We previously demonstrated that the seed-borne bacteria of rice may be a useful and sustainable alternative to pesticides to manage seedling rot, but they are limited in terms of variety. Here, we report that another seed-borne bacterium, Pantoea dispersa BB1, protects rice from B. glumae. We screened 72 bacterial isolates from rice seeds of three genetically different cultivars inoculated or non-inoculated with B. glumae. 16S rRNA gene sequencing revealed that pathogen inoculation affected the composition of culturable seed-borne bacterial communities and increased the presence of Pantoea and Paenibacillus species. Among three Pantoea and Paenibacillus isolates that exhibit tolerance to toxoflavin, a virulence factor of B. glumae, P. dispersa BB1 significantly mitigated the symptoms of rice seedling rot. The culture filtrate of BB1 inhibited the growth of B. glumae in vitro, suggesting that this isolate secretes antibacterial compounds. Seed treatment with BB1 suppressed pathogen propagation in plants, although seed treatment with the culture filtrate did not. Because BB1 did not show pathogenicity in rice, our findings demonstrate that BB1 is a promising biocontrol agent against seedling rot

Enhancement of MAMP signaling by chimeric receptors improves disease resistance in plants

Plants activate defense responses through the recognition of microbe-associated molecular patterns (MAMPs). Recently, several pattern-recognition receptors (PRRs) have been identified in plants, paving the way for manipulating MAMP signaling. CEBiP is a receptor for the chitin elicitor (CE) identified in the rice plasma membrane and XA21 is a member of the receptor-like protein kinase (RLK) family that confers disease resistance to rice bacterial leaf blight expressing the sulfated protein Ax21. To improve resistance to rice blast, the most serious fungal disease of rice, we aimed to create a defense system that combines high affinity of CEBiP for CE and the ability of XA21 to confer disease resistance. Cultured rice cells expressing the chimeric receptor CRXA, which consists of CEBiP and the intracellular region of XA21, induced cell death accompanied by an increased production of reactive oxygen and nitrogen species after exposure to CE. Rice plants expressing the chimeric receptor exhibited more resistance to rice blast. Engineering PRRs may be a new strategy in molecular breeding for achieving disease resistance

OsCERK1 plays a crucial role in the lipopolysaccharide-induced immune response of rice

Plant cell surface receptor-like kinases (RLKs) mediate the signals from microbe-associated molecular patterns (MAMPs) that induce immune responses. Lipopolysaccharide (LPS), the major constituent of the outer membrane of gram-negative bacteria, is a common MAMP perceived by animals and plants; however, the plant receptors/co-receptors are unknown except for LORE, a bulb-type lectin S-domain RLK (B-lectin SD1-RLK) in Arabidopsis. OsCERK1 is a multifunctional RLK in rice that contains lysin motifs (LysMs) and is essential for the perception of chitin, a fungal MAMP, and peptidoglycan, a bacterial MAMP. Here, we analyzed the relevance of OsCERK1 to LPS perception in rice. Using OsCERK1-knockout mutants (oscerk1), we evaluated hydrogen peroxide (H2 O2 ) production and gene expression after LPS treatment. We also examined the LPS response in knockout mutants for the B-lectin SD1-RLK genes in rice and for all LysM-protein genes in Arabidopsis. Compared with wild-type rice cells, LPS responses in oscerk1 cells were mostly diminished. By contrast, rice lines mutated in either of three B-lectin SD1-RLK genes and Arabidopsis lines mutated in the LysM-protein genes responded normally to LPS. From these results, we conclude that OsCERK1 is an LPS receptor/co-receptor and that the LPS perception systems of rice and Arabidopsis are significantly different