A Robust Functional Genomics Approach to Identify Effector Genes Required for Thrips (Frankliniella occidentalis) Reproductive Performance on Tomato Leaf Discs

Abstract

Thrips (Frankliniella occidentalis) is a persistent plant pest that is able to vector pathogenic viruses. Natural plant resistance to thrips has become a prominent breeding target in commercial crops. The main reason for this is the shift toward banning key pesticides used for controlling thrips infestations and the lack of effective alternatives. Despite this urgent need for crop plants that are resistant, or tolerant, to thrips infestation, the toolbox for studying genetic resistance to this insect is still underdeveloped. Essentially, there is a lack of robust protocols for the screening and identification of thrips genes relevant to its performance on crop plants. To bridge this gap, we have developed a functional analysis screening method. Our approach relies on the, Agrobacterium tumefaciens-mediated, homogeneous, and transient ectopic expression of thrips genes in large tomato leaf discs followed by the assessment of thrips reproductive performance. The setup is designed to maintain gene expression during the course of the assay, where GFP signal in the control treatment is used as a reporter of expression. The screen is conducted in a climate box under controlled settings. As a result, multiple genes can be screened for their effect on thrips reproductive performance in a single experiment and in a relatively small space, without the need for generating stable transgenic plants. The method also eliminates the need for a greenhouse equipped to accommodate the combination of A. tumefaciens-infiltrations and thrips infestations. It is not only flexible and convenient for screening genes encoding putative thrips effectors but also for plant resistance genes or effector-targets of host plants and can be adapted for other crop plants, or other herbivorous arthropods.

Keywords: effectors; functional-genomics; resistance; thrips bioassay; tomato.

Figure 1

Setup thrips rearing, vacuum infiltration and plate assay. (A) thrips rearing box with an open lid that has been sealed with mesh, (B) a 50 ml syringe converted to a vacuum infiltration device with a ventilation hole (arrow), (C) leaf discs are lifted away from the agar from one side using tube coders (arrows), and (D) modified 3.5 cm ø 6-well plate caps with an open top sealed with mesh.

Figure 2

Thrips collection and starvation setup and the effect of agro-infiltration on leaf-disc phenotype. (A) Small size plastic hose covered at one end with mesh and a 1 ml pipette tip used to capture thrips by sucking. (B) Thrips starvation setup consisting of a plastic box in which a small water container has been placed, covered with a stretched layer of Parafilm. The lid of the box contains an opening that has been covered with mesh. (C) Tomato leaf discs infiltrated with A. tumefaciens strain 1D1249 carrying an in planta expression construct at 1 dpi or at 8 dpi (D).

Figure 3

GFP fluorescence signal on whole leaf discs of Nicotiana benthamiana at 5 dpi and the phenotype of the GFP signal. (A) N. benthamiana leaf disc infiltrated with buffer (left) or with A. tumefaciens strain GV3101 carrying pTRBO-FLAG-GW (right) at 5 dpi. (B) Microscopic image of GFP signal from the leaf disc in (A). (C,D) GFP signal phenotype observed as a result of hypoxia when the leaf discs were not sufficiently recovered from the vacuum infiltration of A. tumefaciens in N. benthamiana (C) or tomato (D).

Figure 4

Successful expression of GFP in tomato leaf discs after vacuum infiltration with Agrobacterium tumefaciens and recovery from infiltration. Leaf discs prepared from 3.5 to 4-week-old tomato plants of the cultivar Moneymaker were vacuum-infiltrated with the Agrobacterium tumefaciens strain 1D1249 carrying the plasmid pGWB512 in which the eGFP is expressed under the control of the 35S promoter of cauliflower mosaic virus. Leaf discs were allowed to recover from the infiltration for 1 h under an air stream and then placed on water agar in 6-well plates. Pictures were taken using a wide-field fluorescence microscope at the indicated time points and magnification.

Figure 5

Effect of transient expression of thrips effector-candidate genes on thrips reproductive performance. The box plot represents the results from an effector screen, showing the number of nymphs scored at 8 days post-agro-infiltration to transiently express eGFP (control) or thrips effector candidate genes in tomato leaf discs. Oviposition by adult female thrips was started at 2 dpi and continued for 24 h. Mock represents leaf discs infiltrated with only infiltration medium. The box center-lines represent the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; outliers are represented by detached dots; crosses represent sample means; data points are plotted as open circles; n is between 6 and 12 leaf discs. ^*LSD, P = 0.07.