Inducible transgenic tobacco system to study the mechanisms underlying chlorosis mediated by the silencing of chloroplast heat shock protein 90

Abstract

Chlorosis is one of the most common symptoms of plant diseases, including those caused by viruses and viroids. Recently, a study has shown that Peach latent mosaic viroid (PLMVd) exploits host RNA silencing machinery to modulate the virus disease symptoms through the silencing of chloroplast-targeted heat shock protein 90 (Hsp90C). To understand the molecular mechanisms of chlorosis in this viroid disease, we established an experimental system suitable for studying the mechanism underlying the chlorosis induced by the RNA silencing of Hsp90C in transgenic tobacco. Hairpin RNA of the Hsp90C-specific region was expressed under the control of a dexamethasone-inducible promoter, resulted in the silencing of Hsp90C gene in 2 days and the chlorosis along with growth suppression phenotypes. Time course study suggests that a sign of chlorosis can be monitored as early as 2 days, suggesting that this experimental model is suitable for studying the molecular events taken place before and after the onset of chlorosis. During the early phase of chlorosis development, the chloroplast-and photosynthesis-related genes were downregulated. It should be noted that some pathogenesis related genes were upregulated during the early phase of chlorosis in spite of the absence of any pathogen-derived molecules in this system.

Inducible expression of magnesium protoporphyrin chelatase subunit I (CHLI)-amiRNA provides insights into cucumber mosaic virus Y satellite RNA-induced chlorosis symptoms

“…8b). Although the induction of PR4 was observed in iTav-3 in our previous reports [5,42], it was not observed in this study for some reason, suggesting that the induction of ethylene-responsive genes in iTav-3 could be affected by some growth condition. Because we observed clear chlorosis in iTav-3 in both previous and present study, the induction of PR4 gene expression is unlikely to be crucial for the induction of chlorosis.…”

“…The present experimental system has an advantage over the virus-infected plants, mutants, and the constitutively silenced transgenic plants in that it enables us to analyze early phase of chlorosis development, as manifested by the decreased chlorophyll content, the downregulation of CPRGs expression, and the induction of defense-related genes at 2 dpt, when the plants had not shown any visible sign of chlorosis development. As with the previously reported inducible chlorosis systems, iTav tobacco [42] and Hsp90C-hpRNA transgenic plants [5], the present system would also serve as a promising tool to analyze precise mechanisms leading to leaf chlorosis. In this study, we selected two transgenic lines, CHLI-1-11 and CHLI-2-8.…”

“…Also, our recent study has demonstrated the drastic upregulation of PR1a gene during the chlorosis development in Hsp90C silenced transgenic tobacco [5]. To investigate whether it is the case in the present inducible chlorosis system with inducible CHLI silencing, we analyzed the expression of salicylic acid (SA)-responsive PR1a, ethylene-responsive PR4 [6], and jasmonic acid (JA)-responsive SAMDC [28] at 2 dpt.…”

“…Threeweek-old T 2 seedlings from these transgenic lines, nontransformed SR1, transformed control Hsp90C-6 [5], and iTav-3 lines were treated with or without 50 lM Dex. The expression analysis for CHLI-transcripts was performed with samples from 2 dpt.…”

“…To this end, we employed artificial microRNAs (amiRNAs) designed to target CHLI mRNA and chemically inducible promoter. In this system, chlorosis could be induced in transgenic tobacco plants as in the previously reported inducible chlorosis systems, in which CaMV Tav [41,42] and hairpin RNA of Hsp90C [5] are expressed in a chemical-inducible manner. The present system enabled us to analyze early events and temporary changes therein, revealing that CHLI silencing did not only resulted in the decreased chlorophyll content but also caused downregulation of CPRGs expression.…”