Mitogen-activated protein kinase (MPK) cascades play vital roles in plant innate immunity, growth, and development. Here, we report that the rice (Oryza sativa) transcription factor gene OsWRKY31 is a key component in a MPK signaling pathway involved in plant disease resistance in rice. We found that the activation of OsMKK10-2 enhances resistance against the rice blast pathogen Magnaporthe oryzae and suppresses growth through an increase in jasmonic acid and salicylic acid accumulation and a decrease of indole-3-acetic acid levels. Knockout of OsWRKY31 compromises the defense responses mediated by OsMKK10-2. OsMKK10-2 and OsWRKY31 physically interact, and OsWRKY31 is phosphorylated by OsMPK3, OsMPK4, and OsMPK6. Phosphomimetic OsWRKY31 has elevated DNA-binding activity and confers enhanced resistance to M. oryzae. In addition, OsWRKY31 stability is regulated by phosphorylation and ubiquitination via RING-finger E3 ubiquitin ligases interacting with WRKY 1 (OsREIW1). Taken together, our findings indicate that modification of OsWRKY31 by phosphorylation and ubiquitination functions in the OsMKK10-2-mediated defense signaling pathway.

Summary: Rice blast disease, caused by the fungus Magnaporthe oryzae, is the most devastating disease of rice. In our ongoing characterization of the defence mechanisms of rice plants against M. oryzae, a terpene synthase gene OsTPS19 was identified as a candidate defence gene. Here, we report the functional characterization of OsTPS19, which is up‐regulated by M. oryzae infection. Overexpression of OsTPS19 in rice plants enhanced resistance against M. oryzae, while OsTPS19 RNAi lines were more susceptible to the pathogen. Metabolic analysis revealed that the production of a monoterpene (S)‐limonene was increased and decreased in OsTPS19 overexpression and RNAi lines, respectively, suggesting that OsTPS19 functions as a limonene synthase in planta. This notion was further supported by in vitro enzyme assays with recombinant OsTPS19, in which OsTPS19 had both sesquiterpene activity and monoterpene synthase activity, with limonene as a major product. Furthermore, in a subcellular localization experiment, OsTPS19 was localized in plastids. OsTPS19 has a highly homologous paralog, OsTPS20, which likely resulted from a recent gene duplication event. We found that the variation in OsTPS19 and OsTPS20 enzyme activities was determined by a single amino acid in the active site cavity. The expression of OsTPS20 was not affected by M. oryzae infection. This indicates functional divergence of OsTPS19 and OsTPS20. Lastly, (S)‐limonene inhibited the germination of M. oryzae spores in vitro. OsTPS19 was determined to function as an (S)‐limonene synthase in rice and plays a role in defence against M. oryzae, at least partly, by inhibiting spore germination.

Osmotin promoter binding protein 1 (OPBP1), an AP2/ERF transcription factor of tobacco, has been demonstrated to function in disease resistance and salt tolerance in tobacco. To increase stress tolerant capability of rice, we generated rice plants with an OPBP1 overexpressing construct. Salinity shock treatment with 250 mM NaCl indicated that most of the OPBP1 transgenic plants can survive, whereas the control seedlings cannot. Similar recovery was found by using the seedlings grown in 200 mM NaCl for two weeks. The OPBP1 transgenic and control plants were also studied for oxidative stress tolerance by treatment with paraquat, showing the transgenic lines were damaged less in comparison with the control plants. Further, the OPBP1 overexpression lines exhibited enhanced resistance to infections of Magnaporthe oryzae and Rhizoctonia solani pathogens. Gene expressing analysis showed increase in mRNA accumulation of several stress related genes. These results suggest that expression of OPBP1 gene increase the detoxification capability of rice.

Rice plants contain high basal levels of salicylic acid (SA), but some of their functions remain elusive. To elucidate the importance of SA homeostasis in rice immunity, we characterized four rice SA hydroxylase genes (OsSAHs) and verified their roles in SA metabolism and disease resistance. Recombinant OsSAH proteins catalyzed SA in vitro, while OsSAH3 protein showed only SA 5-hydroxylase (SA5H) activity, which was remarkably higher than that of other OsSAHs that presented both SA3H and SA5H activities. Amino acid substitutions revealed that three amino acids in the binding pocket affected SAH enzyme activity and/or specificity. Knockout OsSAH2 and OsSAH3 (sahKO) genes conferred enhanced resistance to both hemibiotrophic and necrotrophic pathogens, whereas overexpression of each OsSAH gene increased susceptibility to the pathogens. sahKO mutants showed increased SA and jasmonate levels compared to those of the wild type and OsSAH-overexpressing plants. Analysis of the OsSAH3 promoter indicated that its induction was mainly restricted around Magnaporthe oryzae infection sites. Taken together, our findings indicate that SA plays a vital role in immune signaling. Moreover, fine-tuning SA homeostasis through suppression of SA metabolism is an effective approach in studying broad-spectrum disease resistance in rice.

The basidiomycetous fungal genus,Rhizoctonia, can cause severe damage to many plants and is composed of multinucleate, binucleate, and uninucleate species differing in pathogenicity. Here we generated chromosome-scale genome assemblies of the three nuclear types ofRhizoctoniaisolates. The genomic comparisons revealed that the uninucleate JN strain likely arose by somatic hybridization of two binucleate isolates, and maintained a diploid nucleus. Homeolog gene pairs in the JN genome have experienced both decelerated or accelerated evolution. Homeolog expression dominance occurred between JN subgenomes, in which differentially expressed genes show potentially less evolutionary constraint than the genes without. Analysis of mating-type genes suggested thatRhizoctoniamaintains the ancestral tetrapolarity of the Basidiomycota. Long terminal repeat-retrotransposons displayed a reciprocal correlation with the chromosomal GC content in the three chromosome-scale genomes. The more aggressive multinucleate XN strain had more genes encoding enzymes for host cell wall decomposition. These findings demonstrate some evolutionary changes of a recently derived hybrid and in multiple nuclear types ofRhizoctonia.

WRKY transcription factors (TFs) have evolved as a plant-specific gene family. Growing evidences indicate that WRKY TFs play crucial roles in plant growth, development, and responses to environmental stimuli. These TFs primarily recognize W-box cis-elements and to a less extent, WT-box. The binding affinity of WRKY TFs to these cis-elements is modulated by protein folding, post-translational modifications, and the nucleotide sequence adjacent to the core motif, including its methylation status. The interaction of WRKY proteins with receptors or as components of receptor complexes offers a potential shortcut signaling pathway for prompt and appropriate responses. Recent studies demonstrated that WRKY TFs can be targeted by effectors from pathogens and insects, leading to shared signaling events between these distinct invaders. Modifications of WRKY TFs by kinases or other regulators can alter their DNA-binding and/or transactivation abilities, thereby impacting the expression of target genes. Additionally, the formation of complexes involving WRKY TFs with other WRKY proteins or components provides valuable insights into the regulatory networks governed by this TF family. This review highlights recent advancements in understanding the interactions between WRKY TFs and other proteins or cis-elements, as well as their roles in responses to biotic and abiotic stresses, metabolism, growth, and development.