Studies on fungal secreted proteins that activate plant immunity in Colletotrichum species

概要

CHAPTER I: Comparative transient expression of two conserved Colletotrichum orbiculare effectors reveal their distinct cell death-inducing activities between Nicotiana benthamiana and melon

Colletotrichum orbiculare infects cucurbits, such as cucumber and melon, as well as the model Solanaceae plant Nicotiana benthamiana, by secreting an arsenal of effectors that suppress the immunity of these distinct plants. Two conserved effectors of C. orbiculare, called NLP1 and NIS1, induce cell death responses in N. benthamiana, suggesting that these secreted effectors activate the plant immunity in N. benthamiana. However, it is unclear whether they exhibit the same activity in Cucurbitaceae plants. In this chapter, I newly established a transient expression system to investigate the cell death-inducing activity of NLP1 and NIS1 in melon (Cucumis melo L.). NLP1 strongly induced cell death in melon but, in contrast to the effects seen in N. benthamiana, mutations in conserved residues in the heptapeptide motif or the putative glycosylinositol phosphorylceramides-binding site of NLP1 did not cancel its cell death-inducing activity in melon. Furthermore, NLP1 lacking the signal peptide caused cell death in melon but not in N. benthamiana. Study of the transient expression of NIS1 also revealed that, unlike in N. benthamiana, NIS1 did not induce cell death in melon. By contrast, NIS1 suppressed flg22-induced ROS generation in melon, as seen in N. benthamiana. These findings indicate distinct cell death- inducing activities of NLP1 and NIS1 in these two plants that C. orbiculare infects.

CHAPTER II: Inappropriate expression of putative pectin-degrading enzymes in multiple Colletotrichum fungi reduces virulence on their host plants via the activation of plant immunity

NLP1 of C. orbiculare is preferentially expressed at the late necrotrophic phase and it was reported that constitutive expression of NLP1 in C. orbiculare impairs pathogen infection on cucurbits via activation of the host plant immunity. In this chapter, I investigated whether other secreted proteins preferentially expressed at the late infection phase have similar effects on pathogen virulence when constitutively expressed. Based on the RNA sequencing data of C. orbiculare, I found that three putative pectate lyases are preferentially expressed at the late infection phase of the pathogen, therefore I decided to focus on the pectate lyases. The studies revealed that constitutive expression of a pectate lyase CoPL3 in C. orbiculare reduced the pathogen virulence toward N.benthamiana but not cucumber. I also revealed that constitutive expression of ChPL3, a C. higginsianum homolog of CoPL3, in C. higginsianum, reduced the pathogen virulence toward multiple brassica plants. Constitutive expression of ChPL3 in C. trifolii also reduced the pathogen virulence toward host plant alfalfa. Mutational studies in ChPL3 also suggested that the enzymatic activity of ChLP3 is involved in ChPL3-dependent negative effects on the virulence of C. higginsianum. Furthermore, I revealed that constitutive expression of two putative pectate lyases (ChPL53 and ChPL85) and a putative pectin lyase (ChPNL73), showing high homology to ChPL3, strongly reduced the virulence of C. higginsianum on a brassica cultivar Komatsuna. These findings revealed that the appropriate expression of multiple secreted proteins including both effectors and cell wall- degrading enzymes is critical for the successful infection of fungal pathogens to avoid the activation of host immune responses.