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A functional genomics approach identifies candidate effectors from the aphid species Myzus persicae (green peach aphid) - PubMed

  • ️Fri Jan 01 2010

A functional genomics approach identifies candidate effectors from the aphid species Myzus persicae (green peach aphid)

Jorunn I B Bos et al. PLoS Genet. 2010.

Abstract

Aphids are amongst the most devastating sap-feeding insects of plants. Like most plant parasites, aphids require intimate associations with their host plants to gain access to nutrients. Aphid feeding induces responses such as clogging of phloem sieve elements and callose formation, which are suppressed by unknown molecules, probably proteins, in aphid saliva. Therefore, it is likely that aphids, like plant pathogens, deliver proteins (effectors) inside their hosts to modulate host cell processes, suppress plant defenses, and promote infestation. We exploited publicly available aphid salivary gland expressed sequence tags (ESTs) to apply a functional genomics approach for identification of candidate effectors from Myzus persicae (green peach aphid), based on common features of plant pathogen effectors. A total of 48 effector candidates were identified, cloned, and subjected to transient overexpression in Nicotiana benthamiana to assay for elicitation of a phenotype, suppression of the Pathogen-Associated Molecular Pattern (PAMP)-mediated oxidative burst, and effects on aphid reproductive performance. We identified one candidate effector, Mp10, which specifically induced chlorosis and local cell death in N. benthamiana and conferred avirulence to recombinant Potato virus X (PVX) expressing Mp10, PVX-Mp10, in N. tabacum, indicating that this protein may trigger plant defenses. The ubiquitin-ligase associated protein SGT1 was required for the Mp10-mediated chlorosis response in N. benthamiana. Mp10 also suppressed the oxidative burst induced by flg22, but not by chitin. Aphid fecundity assays revealed that in planta overexpression of Mp10 and Mp42 reduced aphid fecundity, whereas another effector candidate, MpC002, enhanced aphid fecundity. Thus, these results suggest that, although Mp10 suppresses flg22-triggered immunity, it triggers a defense response, resulting in an overall decrease in aphid performance in the fecundity assays. Overall, we identified aphid salivary proteins that share features with plant pathogen effectors and therefore may function as aphid effectors by perturbing host cellular processes.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Overview of functional genomics pipeline to identify candidate effectors from M. persicae.

(A) Bioinformatics pipeline for data mining of M. persicae salivary gland expressed sequence tags (ESTs). (B) Cloning and functional analyses of candidates to identify effector activities. i) PCR amplification was performed on M. persicae cDNA. ii) Amplicons were cloned in the pCB302-3 vector under control of a 35S promoter and constructs were transformed into Agrobacterium tumefaciens. iii) Multiple clones were sequenced to identify polymorphic candidates. Clones were stored and cultured for subsequent functional assays. iv) Candidate effectors were overexpressed in Nicotiana benthamiana by agroinfiltration to determine whether they induce a phenotype in planta, such as cell death, suppress basal plant defences, PAMP-triggered immunity (PTI), and affect aphid performance.

Figure 2
Figure 2. The candidate effector Mp10 induces chlorosis specifically in N. benthamiana.

(A) Overexpression of 35S-Mp10 by agroinfiltration induces chlorosis in N. benthamiana. Symptoms of chlorosis started to appear from 2 days post infiltration (dpi). Photos were taken 4 dpi. (B) PVX-based expression of Mp10 in N. benthamiana. Symptoms of chlorosis started to appear from 10 days post wound-inoculation (dpwi). Photos were taken 14 dpwi. (C) PVX-based expression of Mp10 in Solanum lycopersicum (tomato). Photos were taken 14 dpwi. D) PVX-based expression of Mp10 in N. tabacum. Photos were taken 14 dpwi. (E) Semi-quantitative RT-PCR on RNA from N. benthamiana (Nb), N. tabacum (Nt) and S. lycopersicum (Sl) plants infected with PVX-Δgfp (vector) or PVX-Mp10 as well as non-infected N. benthamiana plants (Nb, -pvx). Primers were used to amplify sequences corresponding to the PVX virus coat protein (CP) and Mp10. The plant tubulin gene (Tub) was used as a control for equal RNA levels. Plant tissues were harvested 14 dpwi (F) Over-expression of 35S-INF1 and 35S-Mp10 in SGT1-silenced (TRV-SGT1) and control (TRV) N. benthamiana plants. Photos were taken 4 dpi. (G) Percentage of infiltration sites showing either INF1 cell death or Mp10 chlorosis 4 dpi on SGT1-silenced and control N. benthamiana plants. The graphs show the averages calculated from 3 replicated experiments, with 8–10 infiltration sites per individual replicate. Error bars indicate the standard error. H) Semi-quantitative RT-PCR on SGT1-silenced and control N. benthamiana plants with SGT1-specific primers. The plant tubulin gene (Tub) was used as a control for equal RNA amounts.

Figure 3
Figure 3. Mp10 suppresses the oxidative burst induced by flg22, but not chitin, in N. benthamiana.

The induction of reactive oxygen species (ROS) induced by the flg22 and chitin was measured using a luminol-based assay. (A) The ROS response induced by flg22 in N. benthamiana leaf discs overexpressing Mp10, AvrPtoB (positive control) and the vector control upon agroinfiltration. The maximum photon count is based on the average of 8 leaf discs. The experiment was repeated 3 times with similar results. Error bars indicate standard error. Asterisks indicate statistical significance compared to the vector control (p≤0.043) (B) The ROS response induced by chitin in N. benthamiana leaf discs overexpressing Mp10, AvrPtoB (positive control) and the vector control upon agroinfiltration. The maximum photon count is based on the average of 8 leaf discs. The experiment was repeated 3 times with similar results. Error bars indicate standard error. Asterisks indicate statistical significance compared to the vector control (p≤0.028).

Figure 4
Figure 4. A medium-throughput leaf disc-based assay identifies M. persicae effector candidates that affect aphid performance.

(A) A novel medium-throughput assay to determine whether in planta overexpresssion of effector candidates affects aphid performance. i) Effector candidates are overexpressed in Nicotiana benthamiana by agroinfiltration. ii) One day after agroinfiltration leaf discs are harvested from infiltration sites using a cork borer. Leaf discs are placed upside-down on water agar in a 24-wells plate. iii) Four first-instar nymphs are placed on each leaf disc and wells are covered with individual mesh caps. Every six days these four aphids are moved to fresh leaf discs overexpressing the effector candidates. iv) Nymph production is assessed up to 17 days after placing first-instar nymphs on the leaf discs on day 1. (B) Overexpression of Mp10 and Mp42 reduces aphid nymph production (fecundity) and overexpression of MpC002 increases aphid nymph production. For each effector candidate, agroinfiltrations and aphid assays were performed side-by-side with the vector control (vector). Graphs show the average number of nymphs produced per adult based on 3 replicated experiments, each consisting of 6 replicated leaf discs per candidate effector construct (n = 18). Error bars indicate the standard error. Asterisks indicate statistical significance compared to the vector control based on a one-way ANOVA (Mp10: p≤0.026, Mp42: p≤0.036 and MpCOO2: p≤0.038).

Figure 5
Figure 5. Amino acid alignments of M. persicae effector candidates that alter aphid fecundity.

Black lines indicate the predicted signal peptide sequences. (A) Alignment of Mp10 with similar sequences from the aphid species Acyrthosiphon pisum (GenBank accession NP_001119652.1), Megoura viciae (GenBank accession CAG25435.1), and the mosquito species Anopheles gambiae (GenBank accession XP_317401.4). Asterisks indicate conserved cysteine residues. (B) Alignment of Mp42 with a similar sequence from A. pisum (GenBank accession XP_001948510). (C) Alignment of MpC002 with a similar sequence from A. pisum (GenBank accession XP_001948358.1).

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