The mitogen-activated protein kinase (MAPK) signaling pathways regulate diverse cellular processes and have been partially characterized in the rice false smut fungus Ustilaginoidea virens. UvSte50 has been identified as a homolog to Saccharomyces cerevisiae Ste50, which is known to be an adaptor protein for MAPK cascades. ΔUvste50 was found to be defective in conidiation, sensitive to hyperosmotic and oxidative stresses, and non-pathogenic. The mycelial expansion of ΔUvste50 inside spikelets of rice terminated at stamen filaments, eventually resulting in a lack of formation of false smut balls on spikelets. We determined that UvSte50 directly interacts with both UvSte7 (MAPK kinase; MEK) and UvSte11 (MAPK kinase kinase; MEKK), where the Ras-association (RA) domain of UvSte50 is indispensable for its interaction with UvSte7. UvSte50 also interacts with UvHog1, a MAP kinase of the Hog1-MAPK pathway, which is known to have important roles in hyphal growth and stress responses in U. virens. In addition, affinity capture–mass spectrometry analysis and yeast two-hybrid assay were conducted, through which we identified the interactions of UvSte50 with UvRas2, UvAc1 (adenylate cyclase), and UvCap1 (cyclase-associated protein), key components of the Ras/cAMP signaling pathway in U. virens. Together, UvSte50 functions as an adaptor protein interacting with multiple components of the MAPK and Ras/cAMP signaling pathways, thus playing critical role in plant infection by U. virens.

Rice blast, caused by Pyricularia oryzae, is one of the most destructive fungal diseases in rice, severely impacting rice production worldwide every year. Rapid, accurate, and visual detection of P. oryzae is essential for more effective prevention and control. In this study, we developed a recombinase polymerase amplification–lateral flow dipstick (RPA-LFD) assay to detect P. oryzae. Species-specific RPA primer pairs and probe were designed based on the target gene MGG_15975. The optimized reaction temperature and time were set at 37°C and 25 min, respectively. Specificity analysis showed that the assay could specifically detect P. oryzae isolates from rice, whereas other fungal species or Pyricularia species from grasses were not detected. Additionally, this assay demonstrated high sensitivity, capable of detecting as low as 10⁻² ng/μl of P. oryzae genomic DNA, which was found to be 100 times more sensitive than conventional PCR. Furthermore, using this assay, P. oryzae was effectively detected in diseased leaves in rice fields and could also be identified at an early stage of infection before obvious lesions appeared in artificially inoculated rice seedlings. Therefore, the RPA-LFD assay developed in our study for the detection of P. oryzae is rapid, highly sensitive, and efficient, which has the potential application for early diagnosis of P. oryzae infection in rice fields.

Rice false smut (RFS), caused by Villosiclava virens, is an important fungal disease in panicles of rice. V. virens is a heterothallic ascomycete controlled by two opposite idiomorphs, MAT1-1 and MAT1-2. Previous study showed that sexual reproduction of V. virens plays an important role in the epidemic of RFS. In this study, we developed a loop-mediated isothermal amplification (LAMP) assay to detect the mating type of V. virens easily and rapidly by using specific primers based on the mating type genes MAT1-1-2 and MAT1-2-1, respectively. The LAMP assay used only a water/dry bath and could recognize the mating type of V. virens in just 45 min. The LAMP assay was so sensitive that it could detect small amounts of V. virens genomic DNA (as low as 2.0 pg of MAT1-1 and 200.0 pg of MAT1-2) and was 10 times more sensitive than PCR. In addition, we demonstrated the application of mating type via LAMP assay by assessing the genomic DNA of V. virens isolated from rice fields. The high efficiency and specificity of this LAMP assay suggest that it can be used as a rapid testing tool in mating type recognition of V. virens isolates in the field.

Rice false smut (RFS), caused by Ustilaginoidea virens (teleomorph: Villosiclava virens), is a devastating fungal disease that severely impacts global rice production by reducing both yield and grain quality. While the mating-type gene UvMAT1-1-3 is known to regulate both sexual and asexual reproduction in U. virens, its regulatory mechanism remains unclear. In this study, an interacting protein of UvMAT1-1-3, a putative O-methyltransferase (UvPomt), was identified using yeast two-hybrid screening, and its interaction was further confirmed by co-localization microscopy. A quantitative reverse transcription PCR (qRT-PCR) analysis showed a significant up-regulation of UvPomt expression during the early infection stage of U. virens. Functional characterization revealed that ΔUvPomt mutants exhibited reduced fungal pathogenicity, vegetative growth, conidial production, and stress adaptation. Furthermore, a Western blot analysis revealed that the UvMAT1-1-3 protein level was reduced in ΔUvPomt mutants, whereas the UvPomt protein level was elevated in ΔUvMAT1-1-3 mutants. Taken together, these findings suggest a potential reciprocal regulation between UvPomt and UvMAT1-1-3. Understanding UvPomt’s function could provide a potential molecular target for controlling RFS disease.