Localization of Tobacco Yellow Dwarf Virus Replication Using the In Plant Activation (INPACT) Expression Platform

Abstract

Geminiviruses and their diseases are a considerable economic threat to a vast number of crops worldwide. Investigating how and where these viruses replicate and accumulate in their hosts may lead to novel molecular resistance strategies. In this study, we used the Rep-inducible In Plant Activation (INPACT) expression platform, based on the genome of tobacco yellow dwarf virus (TYDV), to determine where this model mastrevirus replicates in its host tobacco. By developing an infectious clone of TYDV and optimizing its delivery by agroinfiltration, we first established an efficient artificial infection process. When delivered into transgenic tobacco plants containing a TYDV-based INPACT cassette encoding the β-glucuronidase (GUS) reporter, we showed the virus activates GUS expression. Histology revealed that reporter gene expression was limited to phloem-associated cell types suggesting TYDV replication has a restricted tissue tropism.

Exploring the source of TYLCV resistance in Nicotiana benthamiana

“…Inoculation of plants with TYLCV and Tomato yellow dwarf virus (TYDV) was conducted on three-weeks old plants using the Agroinfiltration method ( de Felippes and Weigel, 2010 ). The Agrobacterium tumefaciens (strain GV3101) carrying an infectious clone of TYLCV (GenBank GU178815) or TYDV ( Kato et al., 2020 ) at final concentration of OD 600 = 0.1 (in infiltration buffer: MES (20 mM), MgCl 2 (5 mM) and acetosyringone (100 µM)) were used as an inoculum. The TMV-U1 was inoculated mechanically by macerating infected leaf material in 100 mM phosphate buffer and rubbing it on a lower leaf lightly dusted with carborundum.…”