RESUMO
Little is known about the molecular basis of host defense in resistant wild species Zingiber zerumbet (L.) Smith against the soil-borne, necrotrophic oomycete pathogen Pythium myriotylum Drechsler, which causes the devastating soft rot disease in the spice crop ginger (Zingiber officinale Roscoe). We investigated the pattern of host defense between Z. zerumbet and ginger in response to P. myriotylum inoculation. Analysis of gene expression microarray data revealed enrichment of phenylpropanoid biosynthetic genes, particularly lignin biosynthesis genes, in pathogen-inoculated Z. zerumbet compared to ginger. RT-qPCR analysis showed the robust activation of phenylpropanoid biosynthesis genes in Z. zerumbet, including the core genes PAL, C4H, 4CL, and the monolignol biosynthesis and polymerization genes such as CCR, CAD, C3H, CCoAOMT, F5H, COMT, and LAC. Additionally, Z. zerumbet exhibited the accumulation of the phenolic acids including p-coumaric acid, sinapic acid, and ferulic acid that are characteristic of the cell walls of commelinoid monocots like Zingiberaceae and are involved in cell wall strengthening by cross linking with lignin. Z. zerumbet also had higher total lignin and total phenolics content compared to pathogen-inoculated ginger. Phloroglucinol staining revealed the enhanced fortification of cell walls in Z. zerumbet, specifically in xylem vessels and surrounding cells. The trypan blue staining indicated inhibition of pathogen growth in Z. zerumbet at the first leaf whorl, while ginger showed complete colonization of the pith within 36 h post inoculation (hpi). Accumulation of salicylic acid (SA) and induction of SA regulator NPR1 and the signaling marker PR1 were observed in Z. zerumbet. Silencing of PAL in Z. zerumbet through VIGS suppressed downstream genes, leading to reduced phenylpropanoid accumulation and SA level, resulting in the susceptibility of plants to P. myriotylum. These findings highlight the essential role of PAL-dependent mechanisms in resistance against P. myriotylum in Z. zerumbet. Moreover, our results suggest an unconventional role for SA in mediating host resistance against a necrotroph. Targeting the phenylpropanoid pathway could be a promising strategy for the effective management of P. myriotylum in ginger.
Assuntos
Pythium , Zingiber officinale , Zingiberaceae , Pythium/genética , Fenilalanina Amônia-Liase/genética , Fenilalanina Amônia-Liase/farmacologia , Lignina , Ácido Salicílico/farmacologia , Zingiberaceae/genéticaRESUMO
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.