A procedure for the transient expression of genes by agroinfiltration above the permissive threshold to study temperature‐sensitive processes in plant–pathogen interactions

Abstract

Localized expression of genes in plants from T-DNAs delivered into plant cells by Agrobacterium tumefaciens is an important tool in plant research. The technique, known as agroinfiltration, provides fast, efficient ways to transiently express or silence a desired gene without resorting to the time-consuming, challenging stable transformation of the host, the use of less efficient means of delivery, such as bombardment, or the use of viral vectors, which multiply and spread within the host causing physiological alterations themselves. A drawback of the agroinfiltration technique is its temperature dependence: early studies have shown that temperatures above 29 °C are nonpermissive to tumour induction by the bacterium as a result of failure in pilus formation. However, research in plant sciences is interested in studying processes at these temperatures, above the 25 °C experimental standard, common to many host-environment and host-pathogen interactions in nature, and agroinfiltration is an excellent tool for this purpose. Here, we measured the efficiency of agroinfiltration for the expression of reporter genes in plants from T-DNAs at the nonpermissive temperature of 30 °C, either transiently or as part of viral amplicons, and envisaged procedures that allow and optimize its use for gene expression at this temperature. We applied this technical advance to assess the performance at 30 °C of two viral suppressors of silencing in agropatch assays [Potato virus Y helper component proteinase (HCPro) and Cucumber mosaic virus 2b protein] and, within the context of infection by a Potato virus X (PVX) vector, also assessed indirectly their effect on the overall response of the host Nicotiana benthamiana to the virus.

High Temperature, High Ambient CO2 Affect the Interactions between Three Positive-Sense RNA Viruses and a Compatible Host Differentially, but not Their Silencing Suppression Efficiencies

“…A GFP reporter and viral proteins were transiently expressed in plants from caulimovirus 35SP-driven, pROK2-based binary vectors: a vector expressing a free GFP reporter described in [ 26 ]; the binary vector expressing PVY P1-6x-HCPro (construct P1-6x-HCPro), described in [ 27 ]. The binary vector that expressed 2b protein with six histidines fused at its N- terminus, described by in [ 16 ].…”

“…Binary vector pgR107 kindly provided by Prof. D. C. Baulcombe group (University of Cambridge, UK) was used to express a modified infectious PVX that contains an additional CP promoter and a polylinker for the insertion and expression of foreign genes [ 28 ]. PVX expressing PVY P1-6x-HCPro was described in [ 27 ] and PVX expressing Fny CMV 2b protein with 6 histidines fused at its N terminus and an HA peptide at its C- terminus was described in [ 16 ].…”

“…For transient expression of proteins in plants by agroinfiltration, the corresponding binary constructs were transferred to non-oncogenic Agrobacterium tumefaciens strain C58C1, grown and infiltrated as described [ 16 ]. In the case of infectious PVX constructs, the binaries expressing them were expressed from A .…”

“…Recently, two viral suppressors, HCPro from PVY and the 2b protein from the cucumovirus Cucumber mosaic virus (CMV), both ssRNA+ viruses were shown to suppress in N . benthamiana agroinfiltration patch assays the partial silencing of a green fluorescent protein (GFP) reporter at high temperature (30°C) [ 16 ].…”

Virus infection induces resistance to Pseudomonas syringae and to drought in both compatible and incompatible bacteria–host interactions, which are compromised under conditions of elevated temperature and CO2 levels

“…Two-week-old A. thaliana seedlings were agro-infiltrated in vitro with the indicated binary vectors as described [35]. In those cases where expression was to take place at higher temperatures (CCC), plants were maintained during the first 24 h after infiltration at 2 3 °C to allow for the Agrobacterium-mediated T-DNA transfer into plant tissue s to take place, as described [36]. For drought experiments, A. thaliana seedlings were transferred to 5 X 5 cm individual pots containing a mixture of soil and vermiculite (3:1 [v/v]) at 8 days after agro-infiltration (dai) with viruses.…”