Black pepper (Piper nigrum L.), a tropical spice crop of global acclaim, is susceptible to Phytophthora capsici, an oomycete pathogen which causes the highly destructive foot rot disease. A systematic understanding of this phytopathosystem has not been possible owing to lack of genome or proteome information. In this study, we explain an integrated transcriptome-assisted label-free quantitative proteomics pipeline to study the basal immune components of black pepper when challenged with P. capsici. We report a global identification of 532 novel leaf proteins from black pepper, of which 518 proteins were functionally annotated using BLAST2GO tool. A label-free quantitation of the protein datasets revealed 194 proteins common to diseased and control protein datasets of which 22 proteins showed significant up-regulation and 134 showed significant down-regulation. Ninety-three proteins were identified exclusively on P. capsici infected leaf tissues and 245 were expressed only in mock (control) infected samples. In-depth analysis of our data gives novel insights into the regulatory pathways of black pepper which are compromised during the infection. Differential down-regulation was observed in a number of critical pathways like carbon fixation in photosynthetic organism, cyano-amino acid metabolism, fructose, and mannose metabolism, glutathione metabolism, and phenylpropanoid biosynthesis. The proteomics results were validated with real-time qRT-PCR analysis. We were also able to identify the complete coding sequences for all the proteins of which few selected genes were cloned and sequence characterized for further confirmation. Our study is the first report of a quantitative proteomics dataset in black pepper which provides convincing evidence on the effectiveness of a transcriptome-based label-free proteomics approach for elucidating the host response to biotic stress in a non-model spice crop like P. nigrum, for which genome information is unavailable. Our dataset will serve as a useful resource for future studies in this plant. Data are available via ProteomeXchange with identifier PXD003887.

Zingiber zerumbet (Zingiberaceae) is a wild, tropical medicinal herb that shows a high degree of resistance to diseases affecting cultivated ginger. Barley stripe mosaic virus (BSMV) silencing vectors containing an endogenous phytoene desaturase (PDS) gene fragment were agroinfiltrated into young leaves of Z. zerumbet under controlled growth conditions to effect virus-induced gene silencing (VIGS). Infiltrated leaves as well as newly emerged leaves and tillers showed visual signs of PDS silencing after 30 days. Replication and systemic movement of the viral vectors in silenced plants were confirmed by RT-PCR. Real-time quantitative PCR analysis verified significant down-regulation of PDS transcripts in the silenced tissues. Label-free proteomic analysis was conducted in leaves with established PDS transcript down regulation and buffer-infiltrated (mock) leaves. A total of 474 proteins were obtained, which were up-regulated, down-regulated or modulated de novo during VIGS. Most of these proteins were localized to the chloroplast, as revealed by UniprotKB analysis, and among the up-regulated proteins there were abiotic stress responsive, photosynthetic, metabolic and membrane proteins. Moreover, the demonstration of viral proteins together with host proteins proved successful viral infection. We report for the first time the establishment of a high-throughput gene functional analysis platform using BSMV-mediated VIGS in Z. zerumbet, as well as proteomic changes associated with VIGS.

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Piper nigrum L. (black pepper), a woody perennial spice crop indigenous to India is positioned at the phylogenetically unique basal lineage of angiosperms. Cultivation of this major spice crop is constrained by rampant fungal and viral infections leading to a lack of disease-free planting material. The major disease that poses severe threat to P. nigrum plantations and nurseries is ‘quick wilt’ caused by the oomycete Phytophthora capsici, which affects the leaf, stem, spike, collar and root. In this paper, we report the consequence of priming in modulating Piper nigrum defense against Phytophthora capsici. Glycol Chitosan (GC) was used to infiltrate detached leaves of mature P. nigrum plants. It was observed that pre-treatment of GC for 24 hours resulted in significant reduction of disease symptoms in infected leaves, as evidenced by the marked decrease in the size of lesions, and also delayed the appearance of symptoms up to 72 hpi. Experiments repeated in P. nigrum seedlings under controlled growth conditions indicate that delayed disease symptoms of GC pre-treated leaves do not spread to healthy uninfiltrated leaves suggesting a priming-associated systemic defense response. An ROS-mediated manifestation of Hypersensitive Response (HR) induced by Chitosan was also evident in pre-treated leaves. A corresponding visual indication of increased lignification was observed, which correlated with an enhanced lignin content of GC-treated leaves. Enhanced callose deposition was also apparent in GC infiltrated leaves, establishing a stimulatory effect of GC in triggering HR through ROS production, enhanced lignification and callose deposition. Key genes of the core phenylpropanoid and isoprenoid pathways along with major defense signalling pathway genes of P. nigrum, including pathogenesis-related genes and hormone signalling genes showed significant transcript enrichment consequential to GC treatment. A significant quantitative enhancement in Piperine content was evident in GC-infiltrated leaves. The systemic nature of priming on disease protection was established through experiments conducted in rooted cuttings monitored for 30 days after disease infection. This is the first report that provides strong molecular evidence endorsing the twofold advantage of defense priming in P. nigrum by improving crop protection with a concomitant enhancement in Piperine biosynthesis.

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