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Review
. 2013 Mar 22;5(3):998-1022.
doi: 10.3390/v5030998.

Discovering host genes involved in the infection by the Tomato Yellow Leaf Curl Virus complex and in the establishment of resistance to the virus using Tobacco Rattle Virus-based post transcriptional gene silencing

Henryk Czosnek  1 , Assaf Eybishtz, Dagan Sade, Rena Gorovits, Iris Sobol, Eduardo Bejarano, Tábata Rosas-Díaz, Rosa Lozano-Durán
Affiliations
Review

Discovering host genes involved in the infection by the Tomato Yellow Leaf Curl Virus complex and in the establishment of resistance to the virus using Tobacco Rattle Virus-based post transcriptional gene silencing

Henryk Czosnek et al. Viruses. .

Abstract

The development of high-throughput technologies allows for evaluating gene expression at the whole-genome level. Together with proteomic and metabolomic studies, these analyses have resulted in the identification of plant genes whose function or expression is altered as a consequence of pathogen attacks. Members of the Tomato yellow leaf curl virus (TYLCV) complex are among the most important pathogens impairing production of agricultural crops worldwide. To understand how these geminiviruses subjugate plant defenses, and to devise counter-measures, it is essential to identify the host genes affected by infection and to determine their role in susceptible and resistant plants. We have used a reverse genetics approach based on Tobacco rattle virus-induced gene silencing (TRV-VIGS) to uncover genes involved in viral infection of susceptible plants, and to identify genes underlying virus resistance. To identify host genes with a role in geminivirus infection, we have engineered a Nicotiana benthamiana line, coined 2IRGFP, which over-expresses GFP upon virus infection. With this system, we have achieved an accurate description of the dynamics of virus replication in space and time. Upon silencing selected N. benthamiana genes previously shown to be related to host response to geminivirus infection, we have identified eighteen genes involved in a wide array of cellular processes. Plant genes involved in geminivirus resistance were studied by comparing two tomato lines: one resistant (R), the other susceptible (S) to the virus. Sixty-nine genes preferentially expressed in R tomatoes were identified by screening cDNA libraries from infected and uninfected R and S genotypes. Out of the 25 genes studied so far, the silencing of five led to the total collapse of resistance, suggesting their involvement in the resistance gene network. This review of our results indicates that TRV-VIGS is an exquisite reverse genetics tool that may provide new insights into the molecular mechanisms underlying plant infection and resistance to infection by begomoviruses.

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Figures

Figure 1
Figure 1
Generation and phenotypic analysis of TYLCSV-infected 2IRGFP N. benthamiana transgenic plants. a. Construct 2IRGFP contains a direct repeat of the TYLCSV IR encompassing a GFP expression cassette that contains the 35S CaMV promoter (P35S), the complete ORF of GFP and the NOS terminator (Ter). During TYLCSV infection, the viral Rep protein specifically recognizes the IRs flanking the cassette, and mGFP replicons are generated (EM), which in turn leads to a strong over-expression of the GFP transgene and the subsequent accumulation of the fluorescent protein. b. Evolution of virus replication-associated phenotype (RAP) in infected 2IRGFP plants at different days post-infection (dpi). A representative photograph of each RAP phenotype showing the extension and intensity of GFP expression is displayed.
Figure 2
Figure 2
Screening of candidate genes in 2IRGFP transgenic N. benthamiana plants. Plants were co-inoculated with a TRV:Gene construct and TYLCSV. GFP expression was monitored daily up to 15 days post-inoculation (dpi). The picture shows GFP expression in one of the apical leaves under UV (left) and visible light (right) of 2IRGFP N. benthamiana transgenic plants 15 days after they were co-infected with TYLCSV and TRV constructs to induced silencing of genes classified in category A (Replication associated protein A, RPA32, and Ubiquitin activating enzyme 1, UBA1) or category B (Coatomer delta subunit, deltaCOP, and Heat shock cognate 70, HSC70). Leaves from control 2IRGFP plants are shown: agroinfiltrated with an empty binary vector (Mock) or with the empty TRV vector (TRV). The relative amount of TYLCSV DNA accumulated in co-infected plants was quantified by qPCR; results are shown below the images. Values are the mean of five to ten plants. The numbers correspond to the mean ±standard error. This experiment was repeated three times with similar results.
Figure 3
Figure 3
Genes preferentially expressed in R plants (Gene ontology, cellular component). The number of genes silenced so-far and the genes which silencing leads to collapse of resistance are indicated.
Figure 4
Figure 4
Relative amounts of transcripts of Permease I, Hexose transporter LeHTe1, and Lipocalin-like genes in R tomato plants (Ro:0), infected R tomato plants (Ri:0) and infected R tomato plants with silenced Permease I (Ri:TRV-Perm), Hexose transporter LeHTe1 (Ri:TRV-Hex), and Lipocalin-like (Ri:TRV-Lip) genes. Tubulin RNA was used as a reference gene transcript for each of the plants analyzed by qPCR. The amount of transcript immediately before silencing (at day 0) is taken as 1. Average of triplicate measures of three different plants. Bars: standard error.
Figure 5
Figure 5
Collapse of resistance in infected R plants where the Permease I gene has been silenced. a: R tomato plants 8 weeks after TYLCV inoculation; Ri:0, not silenced; Ri:TRV-Per, silenced. Note that Ri:0 do not present symptoms and yield fruits, in comparison Ri:TRV-Per are symptomatic and present inhibited growth. b: Relative amounts of virus (measured by qPCR) in infected tomato plants 3 and 28 days after inoculation; Si:0 is S plants, Ri:0 is R plants and Ri:TRV-Per is R plants where the Permease I gene has been silenced. The amount of virus in Ri:0 plants at 28 dpi was considered as 1.
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