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Successful transmission of tomato spotted wilt virus (TSWV) by Frankliniella occidentalis requires robust infection of the salivary glands (SGs) and virus delivery to plants during salivation. Feeding behavior and transmission efficiency are sexually-dimorphic traits of this thrips vector species. Proteins secreted from male and female SG tissues, and the effect of TSWV infection on the thrips SG proteome are unknown. To begin to discern thrips factors that facilitate virus infection of SGs and transmission by F. occidentalis, we used gel- and label-free quantitative and qualitative proteomics to address two hypotheses: (i) TSWV infection modifies the composition and/or abundance of SG-expressed proteins in adults; and (ii) TSWV has a differential effect on the male and female SG proteome and secreted saliva. Our study revealed a sex-biased SG proteome for F. occidentalis, and TSWV infection modulated the SG proteome in a sex-dependent manner as evident by the number, differential abundance, identities and generalized roles of the proteins. Male SGs exhibited a larger proteomic response to the virus than female SGs. Intracellular processes modulated by TSWV in males indicated perturbation of SG cytoskeletal networks and cell-cell interactions (basement membrane, BM and extracellular matrix proteins, ECM), and subcellular processes consistent with a metabolic slow-down under infection. Several differentially-abundant proteins in infected male SGs play critical roles in viral life cycles of other host-virus pathosystems. In females, TSWV modulated processes consistent with tissue integrity and active translational and transcriptional regulation. A core set of proteins known for their roles in plant cell-wall degradation and protein metabolism were identified in saliva of both sexes, regardless of virus infection status. Saliva proteins secreted by TSWV- infected adults indicated energy generation, consumption and protein turnover, with an enrichment of cytoskeletal/BM/ECM proteins and tricarboxylic acid cycle proteins in male and female saliva, respectively. The nonstructural TSWV protein NSs - a multifunctional viral effector protein reported to target plant defenses against TSWV and thrips - was identified in female saliva. This study represents the first description of the SG proteome and secretome of a thysanopteran and provides many candidate proteins to further unravel the complex interplay between the virus, insect vector, and plant host.

Additional file 4. Chemosensory receptor sequences.

Additional file 7. Complete ATP-binding cassette transporter (ABC) and carboxylesterase sequences.

Additional file 1. Supplementary notes, figures and small tables.

Additional file 6. Complete and partial cytochrome p450 (CYP) sequences.

Additional file 5. Neuropeptide and protein hormone peptide sequences with highlighted features.

Citrus greening or Huanglongbing (HLB) is caused by the phloem-limited intracellular Gram-negative bacterium Candidatus Liberibacter asiaticus (CLas). HLB-infected citrus phloem cells undergo structural modifications that include cell wall thickening, callose and phloem protein induction, and cellular plugging. However, very little is known about the intracellular mechanisms that take place during CLas cell-to-cell movement. Here, we show that CLas movement through phloem pores of sweet orange (Citrus sinensis) and grapefruit (Citrus paradisi) is carried out by the elongated form of the bacteria. The round form of CLas is too large to move, but can change its morphology to enable its movement. CLas cells adhere to the plasma membrane of the phloem cells specifically adjacent to the sieve pores. Remarkably, CLas was present in both mature sieve element cells and nucleated nonsieve element cells. The sieve plate plugging structures of host plants were shown to have different composition in different citrus tissues. Callose deposition was the main plugging mechanism in the HLB-infected flush, where it reduced the open space of the pores. In the roots, pores were surrounded by dark extracellular material, with very little accumulation of callose. The expression of CALLOSE SYNTHASE7 and PHLOEM PROTEIN2 genes was upregulated in the shoots, but downregulated in root tissues. In seed coats, no phloem occlusion was observed, and CLas accumulated to high levels. Our results provide insight into the cellular mechanisms of Gram-negative bacterial cell-to-cell movement in plant phloem.

There is increasing evidence that acylsugars deter insect pests and plant virus vectors, including the western flower thrips (WFT), Frankliniella occidentalis (Pergande), vector of tomato spotted wilt virus (TSWV). Acylsugars are sugar-polyesters composed of saturated, un-saturated, and variously branched short and long chain fatty acids (FAs) esterified to a glucose (acylglucose) or sucrose (acylsucrose) moiety. We sought to understand how acylsucrose amount and composition of associated FA profiles interacted to mediate resistance to WFT oviposition and TSWV inoculation on tomato leaves. Towards this goal, we examined WFT oviposition and TSWV inoculation behavior on tomato lines bred to exude varying amounts of acylsucrose in association with diverse FA profiles. Our data show that as acylsucrose amounts increased, WFT egg-laying (oviposition) decreased and TSWV inoculation was suppressed. Western flower thrips also responded to FA profiles that included iC4, iC11, nC12 and nC10 FA. These findings support improving acylsugar-mediated resistance against WFT by breeding tomatoes exuding greater amounts of acylsucrose associated with specific FA profiles. We show that increasing acylsucrose amount output by type IV trichomes and selecting for particular FA profiles through advanced breeding profoundly affects WFT behavior in ways that benefit management of WFT as direct pests and as TSWV vectors.

Tomatoes (Solanum lycopersicum L.) have been bred to exude higher amounts or different types of the specialized plant metabolites, acylsugars, from type IV trichomes. Acylsugars are known to deter several herbivorous insect pests, including the western flower thrips (WFT), Frankliniella occidentalis (Pergande); however, all previous studies investigated the effect of acylsugars on leaves, or acylsugar extracts obtained from leaves. In spite of the WFT predilection for flowers, there is a gap in knowledge about flower defenses against thrips damage. This is especially important in light of their capacity to acquire and inoculate viruses in the genus Orthotospovirus, such as Tomato spotted wilt orthotospovirus (TSWV), in flowers. Therefore, we turned our attention to assessing thrips oviposition differences on flowers of 14 entries, including 8 interspecific hybrids, 5 tomato lines bred for specific acylsugar-related characteristics (type IV trichome densities, acylsugar amount, sugar moiety and fatty acid profile), and a fresh market tomato hybrid, Mt. Spring, which only produces trace amounts of acylsugars. Our results show that the density of the acylsugar droplet bearing type IV trichomes is greatest on sepals, relative to other flower structures, and accordingly, WFT avoids oviposition on sepals in favor of trichome-sparse petals. In concordance with past studies, acylsugar amount was the most important acylsugar-related characteristic suppressing WFT oviposition. Certain acylsugar fatty acids, specifically i-C5, i-C9 and i-C11, were also significantly associated with changes in WFT oviposition. These results support continued breeding efforts to increase acylsugar amounts and explore modifications of fatty acid profile and their roles in deterring thrips oviposition. The finding that acylsugar production occurs and reduces thrips oviposition in tomato flowers will be important in efforts to use acylsugar-mediated resistance to reduce incidence of orthotospoviruses such as TSWV in tomato by deterring virus transmission and development of thrips vector populations in the crop.

Acylsugars are important insect defense compounds produced at high levels by glandular trichomes of the wild tomato, Solanum pennellii. Marker-assisted selection was used to select for plants containing the three AGQTL named AG3QTL, AG4QTL, and AG11QTL from self-pollinated populations derived from an interspecific backcross population of CU071026 x (CU071026 x S. pennellii LA716). High acylglucose-accumulating lines were selected from these populations that possess these three AGQTL and the fewest number of extraneous S. pennellii LA716 introgressions. Incorporation of these three acylglucose QTL in the presence of the five standard S. pennellii introgressions of CU071026 altered acylsugar level and sugar moiety, demonstrating epistatic interactions between the acylglucose QTL on both of these traits. Comparison of the lines generated from the two breeding techniques indicated the three acylglucose QTL are essential but not necessarily sufficient for the production of elevated levels of acylglucose acylsugars. Fine-mapping of AG3QTL, AG4QTL, and AG11QTL resulted in less than 1 Mbp intervals for the locations of AG4QTL and AG11QTL; proposals of the causal genes underlying these acylglucose QTL are discussed. Characterization of the fatty acid profile of lines selected out of the interspecific backcross populations revealed an increase in the proportion of acylsugar n-C10 fatty acid acyl chains, possibly governed by one or more of the three acylglucose QTL. Characterization of the acylsugar profile of acylglucose lines selected from the interspecific backcross populations also demonstrated interactions among the acylglucose QTL to further modulate the diversity of acylsugars accumulated. Evaluation of an acylglucose line and controls against the tomato insect pest Frankliniella occidentalis demonstrated that levels of resistance differed among these lines and that the acylsugars accumulated by the acylglucose line were effective at reducing both F. occidentalis oviposition and incidence of Tomato spotted wilt orthotospovirus. However, of some of the acylglucose lines and hybrids tested against Spodoptera exigua did not indicate differences for larval weight gain and survival.

International audience; Diaprepes abbreviatus (L.) is an important pest of citrus in the USA. Currently, no effective management strategies of D. abbreviatus exist in citriculture, and new methods of control are desperately sought. To protect citrus against D. abbreviatus a transgenic citrus rootstock expressing Bacillus thuringiensis Cyt2Ca1, an insect toxin protein, was developed using Agrobacterium-mediated transformation of 'Carrizo' citrange [Citrus sinensis (L) Osbeck Poncirus trifoliate (L) Raf]. The transgenic citrus root stock expressed the cytolytic toxin Cyt2Ca1 constitutively under the control of a 35S promoter in the transgenic Carrizo citrange trifoliate hybrid including the roots that are the food source of larval D. abbreviatus. The engineered citrus was screened by Western blot and RT-qPCR analyses for cyt2Ca1 and positive citrus identified. Citrus trees expressing different levels of cyt2Ca1 transcripts were identified (Groups A-C). High expression of the toxin in the leaves (109 transcripts/ng RNA), however, retarded plant growth. The transgenic plants were grown in pots and the roots exposed to 3week old D. abbreviatus larvae using no-choice plant bioassays. Three cyt2Ca1 transgenic plants were identified that sustained less root damage belonging to Group B and C. One plant caused death to 43% of the larvae that fed on its roots expressed 8×106cyt2Ca1 transcripts/ng RNA. These results show, for the first time, that Cyt2Ca1 expressed in moderate amounts by the roots of citrus does not retard citrus growth and can protect it from larval D. abbreviatus.