Calcium (Ca²⁺) signaling is one of the earliest factors to coordinate plant adaptive responses. As direct sensors and activators of Ca²⁺ signals, calcium-dependent protein kinases (CDPKs) were reported to be widely involved in regulating different biotic and abiotic stress stimuli. In this study, 32 Hevea brasiliensis CDPK (HbCDPK) genes were predicted and classified into four subgroups. Among them, the full-length coding sequences of 28 HbCDPK genes were confirmed by RT-PCR and verified by sequencing. Putative cis-elements assay in the promoters of HbCDPKs showed that most of the HbCDPK genes contained gibberellic acid-responsive element (GARE), abscisic acid-responsive element (ABRE), salicylic acid-responsive element (SARE), defense and stress responsive element (TC-rich repeats) and low-temperature response element (LTR), which could be activated by different biotic and abiotic stresses. Real-time PCR analysis indicated that 28 HbCDPK genes respond to infection of pathogenic fungi and a variety of phytohormones. Subcellular localization was observed with most HbCDPKs located in cell membrane, cytoplasm or organelles. Some HbCDPKs were confirmed to cause reactive oxygen species (ROS) production and accumulation in rubber tree mesophyll protoplast directly. HbCDPK5 was strongly induced by the inoculation with Colletotrichum gloeosporioides and was chosen for further analysis. HbCDPK5 localized to the cell membrane and cytoplasm, and obviously regulated the accumulation of ROS in rubber tree mesophyll protoplast. Overexpression of HbCDPK5 in Arabidopsis enhanced the resistance to Botrytis cinerea. These results indicate that rubber tree CDPK genes play important roles in plant disease resistance.

Heat shock transcription factors (HSFs) are a family of transcription regulators. Although HSFs’ functions in controlling the transcription of the molecular chaperone heat shock proteins and resistance to stresses are well established, their effects on the pathogenicity of plant pathogenic fungi remain unknown. In this study, we analyze the role of CgHSF1 in the pathogenicity of Colletotrichum gloeosporioides and investigate the underlying mechanism. Failure to generate the Cghsf1 knock-out mutant suggested that the gene is essential for the viability of the fungus. Then, genetic depletion of the Cghsf1 was achieved by inserting the repressive promoter of nitrite reductase gene (PniiA) before its coding sequence. The mutant showed significantly decrease in the pathogenicity repression of appressorium formation, and severe defects in melanin biosynthesis. Moreover, four melanin synthetic genes were identified as direct targets of CgHSF1. Taken together, this work highlights the role of CgHSF1 in fungal pathogenicity via the transcriptional activation of melanin biosynthesis. Our study extends the understanding of fungal HSF1 proteins, especially their involvement in pathogenicity.

Fungal chitin, as a typical microorganism-associated molecular pattern (PAMP), was recognized by plant LysM-containing protein to induce immunity called pattern-triggered immunity (PTI). To successfully infect host plant, fungal pathogens secreted LysM-containing effectors to inhibit chitin-induced plant immunity. Filamentous fungus Colletotrichum gloeosporioides caused rubber tree anthracnose which resulted in serious loss of natural rubber production worldwide. However, little is known about the pathogenesis mediated by LysM effector of C. gloeosporioide. In this study, we identified a two LysM-containing effector in C. gloeosporioide and named as Cg2LysM. Cg2LysM was involved not only in conidiation, appressorium formation, invasion growth and the virulence to rubber tree, but also in melanin synthesis of C. gloeosporioides. Moreover, Cg2LysM showed chitin-binding activity and suppression of chitin-triggered immunity of rubber tree such as ROS production and the expression of defense relative genes HbPR1, HbPR5, HbNPR1 and HbPAD4. This work suggested that Cg2LysM effector facilitate infection of C. gloeosporioides to rubber tree through affecting invasive structure and inhibiting chitin-triggered plant immunity.

The rubber tree (Hevea brasiliensis) is a tropical perennial crop for the primary source of natural rubber. Colletotrichum gloeosporioides from Hevea brasiliensis (C. gloeosporioides Hb) and Colletotrichum acutatum from Hevea brasiliensis (C. acutatum Hb) are the causal agents of rubber tree anthracnose and lead to serious loss of natural rubber production. Inoculation tests showed that C. gloeosporioides Hb possessed higher pathogenicity than C. acutatum Hb to the rubber tree. Genomic analysis revealed that an unknown gene, named CgNPG1 (a Novel Pathogenic Gene 1), was presented in the genome of C. gloeosporioides Hb but not identified in C. acutatum Hb. CgNPG1 was predicted to encode a small secretory protein without any conserved domain. To investigate the functions of CgNPG1 in C. gloeosporioides Hb and in C. acutatum Hb, the gene deletion and overexpression mutants were generated. The phenotype analysis showed that deletion of CgNPG1 led to changed conidia morphology, decreased mycelial growth, conidiation, conidia germination rate, appressorium formation rate, and pathogenicity of C. gloeosporioides Hb to the rubber tree. Meanwhile, heterogeneous expression of CgNPG1 in C. acutatum Hb significantly changed the conidia morphology and improved the mycelial growth rate, conidiation, conidia germination rate, appressorium formation rate, and the pathogenicity of C. acutatum Hb to the rubber tree. Consistently, CgNPG1 increased the expression level of CaCRZ1 and CaCMK1 in C. acutatum Hb. These data suggested that CgNPG1 contributed to mycelial growth, conidiation, the development of invasive structures, and the pathogenicity of Colletotrichum to the rubber tree, which might be related to the modulation of CaCRZ1 and mitogen-activated protein kinase CMK1. Our results provided new insight into CgNPG1 in regulating growth and pathogenicity of the Colletotrichum spp.

Colletotrichum gloeosporioides is widely distributed and causes anthracnose on many crops, resulting in serious economic losses. Common fungal extracellular membrane (CFEM) domain proteins have been implicated in virulence and their interaction with the host plant, but their roles in C. gloeosporioides are still unknown. In this study, a CFEM-containing protein of C. gloeosporioides was identified and named as CgCFEM1. The expression levels of CgCFEM1 were found to be markedly higher in appressoria, and this elevated expression was particularly pronounced during the initial stages of infection in the rubber tree. Absence of CgCFEM1 resulted in impaired pathogenicity, accompanied by notable perturbations in spore morphogenesis, conidiation, appressorium development and primary invasion. During the process of appressorium development, the absence of CgCFEM1 enhanced the mitotic activity in both conidia and germ tubes, as well as compromised conidia autophagy. Rapamycin was found to basically restore the appressorium formation, and the activity of target of rapamycin (TOR) kinase was significantly induced in the CgCFEM1 knockout mutant (∆CgCFEM1). Furthermore, CgCFEM1 was proved to suppress chitin-triggered reactive oxygen species (ROS) accumulation and change the expression patterns of defense-related genes. Collectively, we identified a fungal effector CgCFEM1 that contributed to pathogenicity by regulating TOR-mediated conidia and appressorium morphogenesis of C. gloeosporioides and inhibiting the defense responses of the rubber tree.

Phytopathogenic fungi secretes a range of effectors to manipulate plant defenses. Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) is a soil-borne pathogen that causes destructive banana wilt disease. Understanding the molecular mechanisms behind Foc TR4 effectors and their regulation of pathogenicity is helpful for developing disease control strategies. In the present study, we identified a novel effector, Fusarium special effector 1 (FSE1), in Foc TR4. We constructed FSE1 knock-out and overexpression mutants and investigated the functions of this effector. In vitro assays revealed that FSE1 was not required for vegetative growth and conidiation of Foc TR4. However, inoculation analysis of banana plantlets demonstrated that knock-out of FSE1 increased the disease index, while overexpression of FSE1 decreased it. Microscope analysis suggested that FSE1 was distributed in the cytoplasm and nuclei of plant cells. Furthermore, we identified an MYB transcription factor, MaEFM-like, as the target of FSE1, and the two proteins physically interacted in the nuclei of plant cells. In addition, Transient expression of MaEFM-like induced cell death in tobacco leaves. Our findings suggest that FSE1 is involved in the pathogenicity of Foc TR4 by targeting MaEFM-like.