Previous studies have shown that the high-osmolarity glycerol mitogen-activated protein kinase (HOG MAPK) signaling pathway and its downstream transcription factor CsAtf1 are involved in the regulation of fludioxonil sensitivity in C. siamense. However, the downstream target genes of CsAtf1 related to the fludioxonil stress response remain unclear. Here, we performed chromatin immunoprecipitation sequencing (ChIP-Seq) and high-throughput RNA-sequencing (RNA-Seq) to identify genome-wide potential CsAtf1 target genes. A total of 3809 significantly differentially expressed genes were predicted to be directly regulated by CsAtf1, including 24 cytochrome oxidase-related genes. Among them, a cytochrome P450-encoding gene, designated CsCyp51G1, was confirmed to be a target gene, and its transcriptional expression was negatively regulated by CsAtf1, as determined using an electrophoretic mobility shift assay (EMSA), a yeast one-hybrid (Y1H) assay, and quantitative real-time PCR (qRT-PCR). Moreover, the overexpression mutant CsCYP51G1 of C. siamense exhibited increased fludioxonil tolerance, and the CsCYP51G1 deletion mutant exhibited decreased fludioxonil resistance, which revealed that CsCyp51G1 is involved in fludioxonil sensitivity regulation in C. siamense. However, the cellular ergosterol content of the mutants was not consistent with the phenotype of fludioxonil sensitivity, which indicated that CsCyp51G1 regulates fludioxonil sensitivity by affecting factors other than the ergosterol level in C. siamense. In conclusion, our data indicate that the transcription factor CsAtf1 negatively regulates the cytochrome P450 gene CsCyp51G1 to increase fludioxonil sensitivity in C. siamense.

The plant MYB transcription factor family featured as highly conserved DNA-binding domains consisting of 1 to 4 imperfect repeats (R). Increasing evidence indicates that MYB genes participates in growth, differentiation, metabolism, and biotic and abiotic stress responses. However, the functions of MYB genes in the rubber tree remain to be deeply elucidated, especially R2R3-MYB gene family. In this study, molecular biology, bioinformatics, and qRT-PCR were used to identify and analyze HbR2R3-MYB gene family members in the rubber tree. A total of 132 members of the R2R3-MYB gene family were identified in the rubber tree based on genome-wide level. Most of the HbR2R3-MYBs were mapped to 17 rubber tree chromosomes except four genes. A phylogenetic analysis divided all the HbR2R3-MYBs into 20 subgroups with Arabidopsis thaliana. MEME analysis showed that the protein of HbR2R3-MYBs was characterized by 9 conserved motifs. Twenty-six representative R2R3 HbMYBs from different subgroups were selected for expression profiles analysis and the results revealed that the HbR2R3-MYBs members showed various expression patterns in different tissues, powdery mildew-infected and ethylene treatment, implying the diversity of their functions in rubber trees. These results provide fundamental knowledge for further studying the response of the HbR2R3-MYB family to stress and regulation latex flow in rubber tree.

ATP synthase is a key enzyme in photophosphorylation in photosynthesis and oxidative phosphorylation in respiration, which can catalyze the synthesis of ATP and supply energy to organisms. ATP synthase has been well studied in many animal species but has been poorly characterized in plants. This research identified forty ATP synthase family members in the rubber tree, and the phylogenetic relationship, gene structure, cis-elements, and expression pattern were analyzed. These results indicated that the ATP synthase of mitochondria was divided into three subgroups and the ATP synthase of chloroplast was divided into two subgroups, respectively. ATP synthase in the same subgroup shared a similar gene structure. Evolutionary relationships were consistent with the introns and exons domains, which were highly conserved patterns. A large number of cis elements related to light, phytohormones and stress resistance were present in the promoters of ATP synthase genes in rubber trees, of which the light signal accounts for the most. Transcriptome and qRT-PCR analysis showed that HbATP synthases responded to cold stress and hormone stimulation, and the response to ethylene was most significant. HbMATPR3 was strongly induced by ethylene and salicylic acid, reaching 122-fold and 17-fold, respectively. HbMATP7-1 was 41 times higher than the control after induction by jasmonic acid. These results laid a foundation for further studies on the function of ATP synthase, especially in plant hormone signaling in rubber trees.

Plasma membrane H⁺-ATPases (PMAs) play an important role in the pathogenicity of pathogenic fungi. Lipid droplets are important storage sites for neutral lipids in fungal conidia and hyphae and can be used by plant pathogenic fungi for infection. However, the relationship between plasma membrane H⁺-ATPase, lipid droplets and virulence remains unclear. Here, we characterized a plasma membrane H⁺-ATPase, CsPMA2, that plays a key role in lipid droplet formation, appresorial development and virulence in C. siamense. Deletion of CsPMA2 impaired C. siamense conidial size, conidial germination, appressorial development and virulence but did not affect hyphal growth. ΔCsPMA2 increased the sensitivity of C. siamense to phytic acid and oxalic acid. CsPMA2 was localized to lipids on the plasma membrane and intracellular membrane. Deletion of CsPMA2 significantly inhibited the accumulation of lipid droplets and significantly affected the contents of some species of lipids, including 12 species with decreased lipid contents and 3 species with increased lipid contents. Furthermore, low pH can inhibit CsPMA2 expression and lipid droplet accumulation. Overall, our data revealed that the plasma membrane H⁺-ATPase CsPMA2 is involved in the regulation of lipid droplet formation and affects appressorial development and virulence in C. siamense.