Rice production is severely impacted by pathogens such as Magnaporthe oryzae and the rice stripe virus (RSV). Ineffectiveness in controlling viruses and the excessive use of fungicides have proven traditional chemical pesticides increasingly inadequate. RNA interference (RNAi) represents a cutting-edge approach for combating crop diseases, especially in rice. This study addresses the critical gap in scalable, effective RNAi-based rice disease management by exploring the potential of spray-applied small RNA (sRNA) and double-stranded RNA (dsRNA) molecules. We utilized dsRNAs produced by in vitro transcription and bacterial expression systems and employed layered double hydroxides (LDH) to enhance RNA stability, absorption, and efficacy. Our research demonstrated that modified sRNAs could effectively penetrate M. oryzae cell membranes and inhibit conidial germination and appressorium formation, while LDH-conjugated dsRNAs provided prolonged and enhanced protection against both rice blast and rice stripe diseases. Most importantly, dsRNA treatments resulted in improved agronomic traits or increased crop yields by protecting against blast and stripe diseases. This study also validated the compatibility of these RNA molecules with industrial production methods, highlighting their potential as a scalable and eco-friendly option for managing crop diseases at the gene level. This work not only offers a new direction for rice disease control but also provides a foundation for the broader application of RNAi technology in agricultural pest management.

[Objectives]The purpose of this study was to investigate the inhibitory effect of the in vitro applied small RNAs（sRNA）targeting cytochrome b（Cytb）on the pathogenicity of Magnaporthe oryzae,a causal agent of the rice blast disease. [Methods]The M.oryzae strain Guy11 was incubated with a double stand small RNA(ds-sRNA MoCytb-21 )to assess the inhibitory effect of sRNA on conidial germination and appressorium formation. Both dsRNA MoCytb-500 （treatment）or solution（control）were co-supplied with Guy11 conidia on rice ’Nipponbare’ leaf surface, whereby the inhibitory effect of the in vitro applied sRNA on pathogenicity of Guy11 was examined. [Results]Compared with the control treatments, both conidial germination rates and appressorium formation rates of Guy11 incubated with sRNA were significantly reduced. Structural modifications such as 2′-methoxyl（OMe）and both phosphorothioate and 2′-methoxyl（SOMe）of sRNA could prolong the inhibitory effect. The lesion areas of leaves sprayed with both dsCytb and Guy11 conidia were significantly smaller than the control treatments. [Conclusions]The in vitro application of sRNAs specifically targeting Cytb of M.oryzae can inhibit the pathogenicity of M.oryzae,which has the potential and value to be developed into an efficient and green RNAi fungicide.

Fusarium fungi are the cause of an array of devastating diseases affecting yield losses and accumulating mycotoxins. Fungicides can be exploited against Fusarium and deoxynivalenol (DON) production. However, Fusarium resistance to common chemicals has become a therapeutic challenge worldwide, which indicates that new control agents carrying different mechanisms of action are desperately needed. Here, we found that a nonantibiotic drug, ethylenediaminetetraacetic acid disodium salt (EDTANa(2)), exhibited various antifungal activities against Fusarium species and DON biosynthesis. The infection of wheat seeding caused by F. graminearum was suppressed over 90% at 4 mM EDTANa(2). A similar control effect was observed in field tests. Mycotoxin production assays showed DON production was significantly inhibited, 47% lower than the control, by 0.4 mM EDTANa(2). In vitro experiments revealed a timely inhibition of H2O2 production as quickly as 4 h after amending cultures with EDTANa(2) and the expression of several TRI genes significantly decreased. Chitin synthases of Fusarium were Mn2+-containing enzymes that were strongly inhibited by Mn2+ deficiency. EDTANa(2) inhibited chitin synthesis and destroyed the cell wall and cytomembrane integrity of Fusarium, mainly via the chelation of Mn2+ by EDTANa(2), and thus led to Mn deficiency in Fusarium cells. Taken together, these findings uncover the potential of EDTANa(2) as a fungicide candidate to manage Fusarium head blight (FHB) and DON in agricultural production.

β-tubulin, a component of microtubules, is involved in a wide variety of roles in cell shape, motility, intracellular trafficking and regulating intracellular metabolism. It has been an important fungicide target to control plant pathogen , for example, Fusarium . However, the regulation of fungicide sensitivity by β-tubulin-interacting proteins is still unclear. Here, ASK1 was identified as a β-tubulin interacting protein. The ASK1 regulated the sensitivity of Fusarium to carbendazim (a benzimidazole carbamate fungicide), and multiple cellular processes, such as chromatin separation, conidiation and sexual production. Further, we found the point mutations at 50th and 198th of β 2 -tubulin which caused carbendazim resistance decreased the binding between β 2 -tubulin and ASK1, resulting in the deactivation of ASK1. ASK1, on the other hand, competed with carbendazim to bind to β 2 -tubulin. The point mutation F167Y in β 2 -tubulin broke the intermolecular H-bonds and salt bridges between β 2 -tubulin and ASK1, which reduced the competitive effect of ASK1 to carbendazim and resulted in the similar carbendazim sensitivities in F167Y-ΔASK1 and F167Y. These findings have powerful implications for efforts to understand the interaction among β 2 -tubulin, its interacting proteins and fungicide, as well as to discover and develop new fungicide against Fusarium .

Blast disease is one of the major rice diseases, and causes nearly 30% annual yield loss worldwide. Resistance genes that have been cloned, however, are effective only against specific strains. In cultivation practice, broad-spectrum resistance to various strains is highly valuable, and requires researchers to investigate the basal defense responses that are effective for diverse types of pathogens. In this study, we took a quantitative proteomic approach and identified 634 rice proteins responsive to infections by both Magnaporthe oryzae strains Guy11 and JS153. These two strains have distinct pathogenesis mechanisms. Therefore, the common responding proteins represent conserved basal defense to a broad spectrum of blast pathogens. Gene ontology analysis indicates that the “responding to stimulus” biological process is explicitly enriched, among which the proteins responding to oxidative stress and biotic stress are the most prominent. These analyses led to the discoveries of OsPRX59 and OsPRX62 that are robust callose inducers, and OsHSP81 that is capable of inducing both ROS production and callose deposition. The identified rice proteins and biological processes may represent a conserved rice innate immune machinery that is of great value for breeding broad-spectrum resistant rice in the future.

Exploring the regulatory mechanism played by endogenous rice miRNAs in defense responses against the blast disease is of great significance in both resistant variety breeding and disease control management. We identified rice defense-related miRNAs by comparing rice miRNA expression patterns before and after Magnaporthe oryzae strain Guy11 infection. We discovered that osa-miR164a expression reduced upon Guy11 infection at both early and late stages, which was perfectly associated with the induced expression of its target gene, OsNAC60. OsNAC60 encodes a transcription factor, over-expression of which enhanced defense responses, such as increased programmed cell death, greater ion leakage, more reactive oxygen species accumulation and callose deposition, and upregulation of defense-related genes. By using transgenic rice over-expressing osa-miR164a, and a transposon insertion mutant of OsNAC60, we showed that when the miR164a/OsNAC60 regulatory module was dysfunctional, rice developed significant susceptibility to Guy11 infection. The co-expression of OsNAC60 and osa-miR164a abolished the OsNAC60 activity, but not its synonymous mutant. We further validated that this regulatory module is conserved in plant resistance to multiple plant diseases, such as the rice sheath blight, tomato late blight, and soybean root and stem rot diseases. Our results demonstrate that the miR164a/OsNAC60 regulatory module manipulates rice defense responses to M. oryzae infection. This discovery is of great potential for resistant variety breeding and disease control to a broad spectrum of pathogens in the future.