Yeast two-hybrid and label-free proteomics based screening of maize root receptor to cellulase of Trichoderma harzianum

Abstract

We earlier reported the role of cellulase (Thph1 and Thph2) from Trichoderma harzianum in controlling the Curvularia leaf spot and Fusarium stalk rot through interaction with the maize root. But, the maize root receptor related to cellulase was unknown. Hence, in the present study, we applied label-free proteomics and yeast two-hybrid (Y2H) assay to screen the root receptor that communicates with cellulase by generating a different combination of activation (AD) and binding (bait) domain plasmids such as DDO-SD/-Leu/-Trp QDO/X-SD/-Ade/-His/-Leu/-Trp. The maize roots were treated with T. harzianum strains including knockout Thph1, Thph2 and wild-type and enzyme cellobiohydrolase 1 (CBH1). The treated or untreated maize roots were collected for extraction of RNA and protein for generation of cDNA library and Label-free proteomics assay respectively. The label-free proteomic results indicated that the protein expression in the maize root significantly varied between the treatments (P < 0.05) and the protein clusters were highly expressed in the treatments of T2 (inoculated with one g of conidia of T. harzianum WT Th22), T3 (inoculated with one g of conidia of knockout strains of Thph1 and Thph2), T4 (inoculated with enzyme CBH 1) as compared to T1 (uninoculated control). The Y2H screening results indicated that autophagocytosis associated protein (ZmATG3) and germin-like protein (ZmGLP) from the maize root seemed to communicate with cellulase Thph1 and Thph2 respectively. The present screening results deserve for further cellular localization of ZmATG3 and ZmGLP in the maize root and their single or synergic functioning in induced systemic resistance (ISR) in maize through molecular dialogue between Trichoderma spp. and maize roots.