Studies on virulence-related effectors and transcription factors preferentially expressed at the pre-invasion stage in Colletotrichum orbiculare

概要

Chapter I. Studies on virulence-related effectors preferentially expressed at the preinvasion stage in Colletotrichum orbiculare

Pathogens manipulate effectors to interfere with various defense signaling pathways for successful infection. However, only a small portion of fungal effectors have been characterized for virulence functions, possibly due to the functional redundancy of effectors in virulence. To establish a compatible interaction leading to proliferation, it is plausible that C. orbiculare expresses key virulence-related effectors at a stage when the pathogen has not yet developed invasive hyphae (called the pre-invasion stage) thereby suppressing non-host resistance. To identify C. orbiculare effectors that have a role in the pre-invasion stage, I first selected 8 putative secreted protein genes that are highly expressed in C. orbiculare at 1 dpi on Nicotiana benthamiana, which is considered to be the typical pre-invasion stage. I performed a functional screening of eight candidates via virulence enhancement assay, as a result, the transient expression of SIB1 and SIB2 (Suppression of Immunity in N. benthamiana) significantly enhanced lesion development of C. orbiculare on N. benthamiana. Further studies on the effector SIB1 showed that the generation of reactive oxygen species triggered by two different PAMPs (pathogenassociated molecular patterns), chitin and flg22, were commonly suppressed when SIB1 was transiently expressed in N. benthamiana. In addition to that, transient expression of SIB1 suppressed cell death triggered by INF1 which is a well-known oomycete PAMP elicitor that can induce the hypersensitive response in N. benthamiana leaves. However, the SIB1 knockout mutant of C. orbiculare showed the wild-type virulence on both cucumber and N. benthamiana, indicating that SIB1 is dispensable for virulence of C. orbiculare. This phenotype is possibly due to the functional redundancy of effectors in pathogen virulence. Taking these results together, I here identified one effector called SIB1 that is involved in the suppression of multiple PAMP-triggered immunities.

Chapter II. Studies on virulence-related transcription factors preferentially expressed at the pre-invasion stage in Colletotrichum orbiculare

Colletotrichum fungi secrete a large number of effectors at each different infection stage for successful infection. However, the regulatory mechanism of this stage-specific expression of effectors remains largely unknown. For the timely expression of these effectors for host invasion, host recognition and subsequent signaling are likely important. Then it is plausible that, as a next step, transcription factors play crucial roles in the effectors’ expression. Here I focused on transcription factors highly induced at the preinvasion stage in C. orbiculare on host plants to identify the virulence-related transcription factors of C. orbiculare that mainly control effector expression. The knockout analysis showed that the disruption of TFV1 (transcription factor for virulence 1) reduced the virulence of C. orbiculare against cucurbitaceous plants, including cucumber and melon, but not N. benthamiana. Additional deletion of TVL1 (TFV1 like 1), which is the closest homolog of TFV1 in C. orbiculare, enhanced the reduction of lesion development on cucurbitaceous plants. To investigate the regulatory role of TFV1 and TVL1, comparative RNA sequencing analyses between the wild-type strain and each mutant were performed. The results showed that virulence effector genes (EPC1, EPC2, ECP3, and EPC4) are commonly down-regulated in both tfv1∆ mutant and tfv1∆ tvl1∆ mutant. Furthermore, there is no significant difference in the expression of EPC1, EPC2, ECP3, and EPC4 between single and double knockout mutants, indicating that TFV1 encodes a transcriptional factor critical for the stage-specific EPCs expression. Apart from that, there are 39 putative secreted protein genes specifically downregulated in the tfv1∆ tvl1∆ mutant but not in the tfv1∆ mutant, suggesting that TVL1 contributes to the C. orbiculare virulence probably by controlling the expression of uncharacterized effectors. In conclusion, these results suggest that TFV1 and TVL1 act as two regulators controlling the virulence of C. orbiculare on cucurbitaceous plants through the regulation of virulence effector gene expression.