Functional characterization of Phytophthora parasitica PpRxLR1 and PpRxLR6 effectors in Nicotiana benthamiana

Abstract

Phytophthora parasitica is one of the most destructive oomycetes. During its infection process, P. parasitica secretes hundreds of RxLR effector proteins (including the conserved RxLR effectors (CRE)) into the cytoplasmic region, as putative virulence factors. Remarkable progress has been made in understanding CRE from other Phytophthora spp. However, little is known about CRE from P. parasitica. The aim of this study was to assess the functions of two CRE from P. parasitica. Towards this end, in silico analyses revealed that P. parasitica INRA 310 secrete 71 conserved RxLR effectors. Among the 71 CRE, PpRxLR1 and PpRxLR6 effector proteins were selected for further functional characterization. Inoculation of Nicotiana benthamiana leaves with zoospores indicated that the life cycle of P. parasitica switches from asymptomatic to symptomatic phase. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses of the inoculated leaves showed that P. parasitica PpRxLR1 and PpRxLR6 are expressed during the biotrophic phase, suggesting their important in virulence. Findings from Agrobacterium tumeficiens-mediated transiet expression of PpRxLR1 and PpRxLR6 in N. benthamiana revealed potential mechanisms of P. parasitica PpRxLR1 and PpRxLR6 in promoting disease development, this includes inducing reactive oxygen species as well as callose deposition. In addition, RT-PCR analyses revealed that PpRxLR1 and PpRxLR6 induce phytohormones (SA, ET, JA) and MAPKs (MPK3 and MPK6). These data indicate that both PpRxLR1 and PpRxLR6 are an important virulence factors of P. parasitica. Therefore, functional characterization of CRE from P. parasitica is a promising route for the search of potential durable resistance breeding in plants.