Virus-induced gene silencing (VIGS) for elucidation of pathogen defense role of serine/threonine protein kinase in the non-model plant Piper colubrinum Link.

Abstract

Piper colubrinum Link., an exotic species, is a wild relative of cultivated black pepper (Piper nigrum L.), and shows high degree of resistance to the oomycete pathogen Phytophthora capsici, which causes the devastating ‘quick wilt’ disease in P. nigrum. Serine/threonine protein kinase (PcSTPK) is a prospective candidate gene identified from P. colubrinum for future genetic improvement of P. nigrum. RACE PCR amplified a full-length PcSTPK of 1.7 kbp which encodes 575 amino acid residues with a calculated molecular weight of 64.45 kDa. PcSTPK forms a part of a distinct clade in phylogeny analysis, sharing higher similarity to receptor-like protein kinase class of Ser/Thr protein kinases. Real-time qRT-PCR showed the enhanced expression of PcSTPK gene upon P. capsici inoculation and the maximum accumulation of STPK transcripts was observed at 12 h post inoculation. A Tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) was established using Phytoene desaturase endogenous gene as a reporter. TRV:PcSTPK VIGS vector was infiltrated into young leaves of P. colubrinum. A time course study revealed that STPK transcript levels were significantly downregulated. Knock down of PcSTPK by VIGS increased the susceptibility to P. capsici infection, as evidenced by the appearance of foliar necrotic lesions and increased proliferation and sporulation of P. capsici on the leaf surface. We report the successful implementation of VIGS as a systemic strategy for defense gene functional validation in P. colubrinum and our data provide direct evidence for the possible role of PcSTPK in modulating antifungal defense response in the plant.