Two homologous Zn2Cys6 transcription factors play crucial roles in host specificity of Colletotrichum orbiculare by controlling the expression of cucurbit-specific virulence effectors

Abstract

Introduction

Plant fungal pathogens impose a huge burden of pressure on agricultural productivity and threaten global food security by causing destructive diseases on plants (Avery et al., 2019), and elucidating their infection mechanisms at the molecular level is therefore essential for addressing this challenge. The ascomycete genus Colletotrichum contains > 190 accepted species and causes anthracnose disease in a wide variety of plants, including numerous economically important crops (Cannon et al., 2012; Dean et al., 2012; O'Connell et al., 2012; Jayawardena et al., 2021). In general, Colletotrichum species exhibit a hemibiotrophic lifestyle in host plant infection: pathogens initially invade but keep host cells alive (the biotrophic phase), and later kill them by developing necrotrophic hyphae as well as secreting toxins and lytic enzymes (the necrotrophic phase) (Münch et al., 2008; Kleemann et al., 2012; O'Connell et al., 2012). Among Colletotrichum species, C. orbiculare infects multiple cucurbitaceous plants, such as cucumber, watermelon, and melon (Kubo & Takano, 2013; Matsuo et al., 2022), and can also infect Nicotiana benthamiana, which is distantly related to cucurbitaceous plants (Takano et al., 2006; Inoue et al., 2023).

During invasion and colonization of host plants, plant pathogenic fungi secrete a suite of effectors (Kale & Tyler, 2011; Bozkurt et al., 2012). Effectors are typically small, secreted cysteine-rich proteins that act by suppressing plant immunity or manipulating host environmental factors (Selin et al., 2016). Genome sequence analyses of C. orbiculare revealed that numerous effector candidate genes are present in this pathogen (Gan et al., 2013). So far, several virulence-related effectors have been identified in C. orbiculare. For example, an effector named NIS1 (necrosis-inducing secreted protein 1) that can induce cell death on N. benthamiana was identified by functional screening of C. orbiculare cDNAs (Yoshino et al., 2012), whereas CoDN3 was described as an effector that suppresses NIS1-induced cell death. Subsequently, it was reported that NIS1 targets the plant immune kinases BAK1 (BRI1-ASSOCIATED RECEPTOR KINASE 1) and BIK1 (BOTRYTIS-INDUCED KINASE 1) to suppress plant immune responses triggered by PAMPs (pathogen-associated molecular patterns) (Irieda et al., 2019). Also, the effector CoMC69 was shown to be required for virulence of C. orbiculare on both cucumber and N. benthamiana (Saitoh et al., 2012).

In addition, secreted protein genes of C. orbiculare exhibit different expression profiles during the infection, that is more effector-like genes are upregulated during the establishment of biotrophy, whereas genes encoding degradative enzymes are upregulated in the necrotrophic phase (Gan et al., 2013; Irieda et al., 2016), suggesting the importance of effectors for the early infection steps. Consistent with this idea, we have recently reported the identification of four effector genes, named EPC1 (effector protein for cucurbit infection 1) to EPC4, that are preferentially expressed during early stages of infection and are required for fungal virulence in the cucurbit hosts cucumber and melon (Inoue et al., 2023). Several plant pathogenic fungi such as Colletotrichum higginsianum and Magnaporthe oryzae display expression profiles of secreted protein genes similar to those of C. orbiculare (O'Connell et al., 2012; Gan et al., 2013; Yan et al., 2023). These findings suggest the importance of infection stage-specific expression of effectors for virulence of fungal pathogens; however, it remains to be elucidated how the expression of effector genes is regulated in these pathogens.

Transcription factors are sequence-specific DNA-binding proteins that are essential for regulating gene expression in various contexts such as development and environmental responses. Thus, it is plausible that plant pathogenic fungi deploy a suitable set of transcription factors for successful establishment of host infection, including those that regulate the expression of key effector genes. Until now, multiple transcription factors required for fungal pathogenicity and development have been identified and characterized in several fungal species including C. orbiculare (John et al., 2021). Regarding transcription factors that regulate the expression of effector genes, it was reported that the transcription factor-encoding gene AbPf2 is involved in controlling the expression of 13 genes encoding hydrolytic enzymes and eight encoding putative effector proteins in Alternaria brassicicola (Cho et al., 2013). More recently, two fungal transcription factors (Moeitf1 and Moeitf2) were identified as required for full virulence of M. oryzae on rice, and Moeitf2 controls the expression of the secreted protein gene T2REP, which is involved in pathogen virulence (Cao et al., 2022).

Currently, it is unknown how the infection-related expression of effector genes, including EPC1–EPC4, is regulated in C. orbiculare. Interestingly, the quadruple mutant of the four EPC genes displayed almost complete loss of virulence on the cucurbit hosts but still maintained full virulence on N. benthamiana (Inoue et al., 2023), suggesting the specific involvement of the four EPC effectors in virulence on the cucurbit hosts. Importantly, further studies revealed that the EPC genes are preferentially expressed after inoculation on cucumber compared with inoculation on N. benthamiana, although the expression profiles of the majority of genes were common to both hosts (Inoue et al., 2023).

In this study, based on RNA-Seq data of C. orbiculare at multiple stages, we selected transcription factor genes that are preferentially expressed in the early infection phase of C. orbiculare as candidates for transcription factors involved in virulence and effector regulation. We then performed knockout analysis of these candidate genes and identified one gene required for full virulence of C. orbiculare on the cucurbit hosts, which was named TFV1 (Transcription Factor for Virulence 1). TFV1 encodes a putative transcription factor containing a Zn₂Cys₆ binuclear cluster DNA-binding domain. Phenotypic analyses of the ∆tfv1 mutant showed that TFV1 is involved in appressorial host invasion into cucurbit hosts but that its deletion does not affect the development of appressoria. The additional deletion of TVL1 (TFV1-like 1), which is the closest homolog of TFV1 in C. orbiculare, in the ∆tfv1 mutant further reduced lesion development on the cucurbit hosts. Importantly, the ∆tfv1 ∆tvl1 double mutants were as virulent on N. benthamiana as the wild-type (WT) strain, suggesting that TFV1 and TVL1 of C. orbiculare are specifically important for the infection of cucurbit hosts. Remarkably, RNA-Seq and reverse transcription quantitative polymerase chain reaction (RT-qPCR) analyses revealed that the expression of all four EPC genes was commonly downregulated in both the ∆tfv1 and ∆tfv1 ∆tvl1 mutants. Furthermore, yeast one-hybrid assays showed that TFV1 exhibits binding activity to the putative promoter sequences of EPC2, EPC3, and EPC4 genes. Therefore, the TFV1-encoded transcription factor is critical for the induced expression of key virulence effector genes in cucumber infection. Further analysis also successfully identified a novel virulence effector gene, named EPC5, containing the LysM domain, whose expression is significantly reduced in the ∆tfv1 ∆tvl1 mutant but not in the ∆tfv1 mutant.