Background During anther development, the tapetum provides essential nutrients and materials for pollen development. In rice, multiple transcription factors and enzymes essential for tapetum development and pollen wall formation have been cloned from male-sterile lines. Results In this study, we obtained several lines in which the MYB transcription factor OsMS188 was knocked out through the CRISPR-Cas9 approach. The osms188 lines exhibited a male-sterile phenotype with aberrant development and degeneration of tapetal cells, absence of the sexine layer and defective anther cuticles. CYP703A3, CYP704B2, OsPKS1, OsPKS2, DPW and ABCG15 are sporopollenin synthesis and transport-related genes in rice. Plants with mutations in these genes are male sterile, with a defective sexine layer and anther cuticle. Further biochemical assays demonstrated that OsMS188 binds directly to the promoters of these genes to regulate their expression. UDT1, OsTDF1, TDR, bHLH142 and EAT1 are upstream regulators of rice tapetum development. Electrophoretic mobility shift assays (EMSAs) and activation assays revealed that TDR directly regulates OsMS188 expression. Additionally, protein interaction assays indicated that TDR interacts with OsMS188 to regulate downstream gene expression. Conclusion Overall, OsMS188 is a key regulator of tapetum development and pollen wall formation. The gene regulatory network established in this work may facilitate future investigations of fertility regulation in rice and in other crop species.

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Photoperiod/temperature-sensitive genic male sterility (P/TGMS) is widely applied for improving crop production. Previous investigations using the reversible male sterile (rvms) mutant showed that slow development is a general mechanism for restoring fertility to P/TGMS lines in Arabidopsis. In this work, we isolated a restorer of rvms-2 (res3), as the male sterility of rvms-2 was rescued by res3. Phenotype analysis and molecular cloning show that a point mutation in UPEX1 l in res3 leads to delayed secretion of callase A6 from the tapetum to the locule and tetrad callose wall degradation. Electrophoretic mobility shift assay and chromatin immunoprecipitation analysis demonstrated that the tapetal transcription factor ABORTED MICRO-SPORES directly regulates UPEX1 expression, revealing a pathway for tapetum secretory function. Early degradation of the callose wall in the transgenic line eliminated the fertility restoration effect of res3. The fertility of multiple known P/TGMS lines with pollen wall defects was also restored by res3. We propose that the remnant callose wall may broadly compensate for the pollen wall defects of P/TGMS lines by providing protection for pollen formation. A cellular mechanism is proposed to explain how slow development restores the fertility of P/TGMS lines in Arabidopsis.

Histone H3K4 methylation is catalysed by the multi-protein complex known as the Set1/COMPASS or MLL/COMPASS-like complex, an element that is highly evolutionarily conserved from yeast to humans. However, the components and mechanisms by which the COMPASS-like complex targets the H3K4 methylation of plant pathogenic genes in fungi remain elusive. Here we present a comprehensive analysis combining biochemical, molecular, and genome-wide approaches to characterize the roles of the COMPASS-like family in Magnaporthe oryzae, a model plant fungal pathogen. We purified and identified six conserved subunits of COMPASS from the rice blast fungus M. oryzae, i.e., MoBre2 (Cps60/ASH2L), MoSpp1 (Cps40/Cfp1), MoSwd2 (Cps35), MoSdc1 (Cps25/DPY30), MoSet1 (MLL/ALL) and MoRbBP5 (Cps50), using an affinity tag on MoBre2. We determined the SPRY domain of MoBre2 can recognize directly with DPY30 domain of MoSdc1 in vitro. Furthermore, we found that deletion of the genes encoding COMPASS subunits of MoBre2, MoSpp1 and MoSwd2 caused similar defects regarding invasive hyphal development and pathogenicity. Genome-wide profiling of H3K4me3 revealed that the it has remarkable co-occupancy at the TSS regions of target genes. Significantly, these target genes are often involved in spore germination and pathogenesis. Decreased gene expression caused by the deletion of MoBre2, MoSwd2 or MoSpp1 gene was highly correlated with decrease in H3K4me3. Taken together, these results suggest that MoBre2, MoSpp1, and MoSwd2 function as a whole COMPASS complex, contributing to fungal development and pathogenesis by regulating H3K4me3-targeted genes in M. oryzae.

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