Cristina Rosa

Herbivore exploits orally secreted bacteria to suppress plant defenses

Significance The role of herbivore-associated microbes in modifying plant defenses has received scant attention. The Colorado potato beetle secretes symbiotic bacteria to wounds to manipulate plant defenses. The bacteria elicit salicylic acid (SA)-regulated defenses, and because SA signaling often negatively cross-talks with jasmonate signaling, plants are unable to fully activate their jasmonate-mediated resistance against the herbivore. From the plants’ perspective, they recognize herbivores not as such, but as microbial threats. We identified the specific bacteria from the beetle secretions and also characterized one of the bacterial effectors responsible for defense suppression. This clever, deceptive strategy for suppressing defenses has not been previously documented. Our results add a significant, unique concept to plant–insect interactions and how herbivores hijack plant defense signaling. Induced plant defenses in response to herbivore attack are modulated by cross-talk between jasmonic acid (JA)- and salicylic acid (SA)-signaling pathways. Oral secretions from some insect herbivores contain effectors that overcome these antiherbivore defenses. Herbivores possess diverse microbes in their digestive systems and these microbial symbionts can modify plant–insect interactions; however, the specific role of herbivore-associated microbes in manipulating plant defenses remains unclear. Here, we demonstrate that Colorado potato beetle (Leptinotarsa decemlineata) larvae exploit bacteria in their oral secretions to suppress antiherbivore defenses in tomato (Solanum lycopersicum). We found that antibiotic-untreated larvae decreased production of JA and JA-responsive antiherbivore defenses, but increased SA accumulation and SA-responsive gene expression. Beetles benefit from down-regulating plant defenses by exhibiting enhanced larval growth. In SA-deficient plants, suppression was not observed, indicating that suppression of JA-regulated defenses depends on the SA-signaling pathway. Applying bacteria isolated from larval oral secretions to wounded plants confirmed that three microbial symbionts belonging to the genera Stenotrophomonas, Pseudomonas, and Enterobacter are responsible for defense suppression. Additionally, reinoculation of these bacteria to antibiotic-treated larvae restored their ability to suppress defenses. Flagellin isolated from Pseudomonas sp. was associated with defense suppression. Our findings show that the herbivore exploits symbiotic bacteria as a decoy to deceive plants into incorrectly perceiving the threat as microbial. By interfering with the normal perception of herbivory, beetles can evade antiherbivore defenses of its host.

Oral Delivery of Double-Stranded RNAs and siRNAs Induces RNAi Effects in the Potato/Tomato Psyllid, Bactericerca cockerelli

The potato/tomato psyllid, Bactericerca cockerelli (B. cockerelli), and the Asian citrus psyllid, Diaphorina citri (D. citri), are very important plant pests, but they are also vectors of phloem-limited bacteria that are associated with two devastating plant diseases. B. cockerelli is the vector of Candidatus Liberibacter psyllaurous (solanacearum), which is associated with zebra chip disease of potatoes, and D. citri is the vector of Ca. Liberibacter asiaticus, which is associated with the Huanglongbing (citrus greening) disease that currently threatens the entire Florida citrus industry. Here we used EST sequence information from D. citri to identify potential targets for RNA interference in B. cockerelli. We targeted ubiquitously expressed and gut-abundant mRNAs via injection and oral acquisition of double-stranded RNAs and siRNAs and were able to induce mortality in recipient psyllids. We also showed knockdown of target mRNAs, and that oral acquisition resulted primarily in mRNA knockdown in the psyllid gut. Concurrent with gene knockdown was the accumulation of target specific ∼ 21 nucleotide siRNAs for an abundant mRNA for BC-Actin. These results showed that RNAi can be a powerful tool for gene function studies in psyllids, and give support for continued efforts for investigating RNAi approaches as possible tools for psyllid and plant disease control.

Gut Microbes Contribute to Nitrogen Provisioning in a Wood-Feeding Cerambycid

ABSTRACT Xylophagous insects often thrive on nutritionally suboptimal diets through symbiotic associations with microbes that supplement their nutritional requirements, particularly nitrogen. The wood-feeding cerambycid Anoplophora glabripennis (Motschulsky) feeds on living, healthy host trees and harbors a diverse gut microbial community. We investigated gut microbial contributions to larval nitrogen requirements through nitrogen fixing and recycling (urea hydrolysis) processes, using a combination of molecular, biochemical, and stable isotope approaches. Genes and transcripts of conserved regions of the urease operon (ureC) and nitrogen fixing (nif) regulon (nifH) were detected in A. glabripennis eggs and larvae from naturally infested logs and from larvae reared on artificial diet. Significant nitrogen fixation and recycling were documented in larvae using 15N2 gas and 15N-urea, respectively. Subsequent 15N-routing of incorporated recycled nitrogen into larval essential and nonessential amino acids was shown for 15N-urea diet-fed larvae. Results from this study show significant gut microbial contributions to this insects metabolic nitrogen utilization through nitrogenous waste product recycling and nitrogen fixation.

Tritrophic Interactions: Microbe-Mediated Plant Effects on Insect Herbivores

It is becoming abundantly clear that the microbes associated with plants and insects can profoundly influence plant-insect interactions. Here, we focus on recent findings and propose directions for future research that involve microbe-induced changes to plant defenses and nutritive quality as well as the consequences of these changes for the behavior and fitness of insect herbivores. Insect (herbivore and parasitoid)-associated microbes can favor or improve insect fitness by suppressing plant defenses and detoxifying defensive phytochemicals. Phytopathogens can influence or manipulate insect behavior and fitness by altering plant quality and defense. Plant-beneficial microbes can promote plant growth and influence plant nutritional and phytochemical composition that can positively or negatively influence insect fitness. Lastly, we suggest that entomopathogens have the potential to influence plant defenses directly as endophytes or indirectly by altering insect physiology.

RNA interference is induced in the glassy winged sharpshooter Homalodisca vitripennis by actin dsRNA

BACKGROUND The glassy winged sharpshooter, Homalodisca vitripennis, is an unusually robust and efficient leafhopper vector of Xylella fastidiosa. X. fastidiosa is the causative agent of Pierces disease, almond scorch, citrus variegated chlorosis and other serious plant diseases. The present study was conducted to establish whether RNA interference (RNAi) was induced in nymphal H. vitripennis that were injected with actin dsRNAs and other dsRNAs. RESULTS A dramatic reduction in target H. vitripennis actin mRNAs and the formation of small interfering RNAs (siRNAs), hallmarks of RNAi, were found following the injection of actin dsRNAs. Quantitative reverse transcription PCR indicated an 80% reduction in actin mRNA levels by 5 days post-injection. Western blot analysis showed a dramatic drop in actin protein levels by 3 days post-injection. Biological effects such as incomplete nymphal-adult ecdysis and > 95% mortality were also found following the injection of fifth-instar nymphs with actin dsRNA. Dramatic reductions in target mRNA levels were also found following the injection of other dsRNAs into fifth-instar H. vitripennis. CONCLUSION The findings indicate that RNAi is induced in post-embryonic leafhoppers by dsRNA. The present system can be used to screen potential gene-silencing targets that can be used for reducing the vector competence of H. vitripennis and other leafhoppers.

Detection of Flavescence Dorée Phytoplasma in Grapevine by Reverse-Transcription PCR

Flavescence dorée (FD) is the most serious phytoplasma disease of grapevine. This report describes a novel method of detecting FD phytoplasma based on reverse-transcription polymerase chain reaction (RT-PCR) on 16S ribosomal RNA (16SrRNA) which will greatly improve mass screening of infected grapevines. A rapid protocol for extracting sap from whole leaves or midveins and successive one-tube amplification by RT-PCR was applied to grapevine samples with or without symptoms collected from different areas of Piedmont (northwestern Italy). Results were compared with those obtained using one of the current diagnostic methods that utilizes nested PCR on phytoplasma DNA-enriched preparations. A Cohens kappa index of 0.76 indicated a substantial agreement between the two sets of results. The RT-PCR method has the advantage of being a rapid, reliable, and sensitive assay for large-scale screening of grapevines.

Essential Amino Acid Supplementation by Gut Microbes of a Wood-Feeding Cerambycid

Abstract Insects are unable to synthesize essential amino acids (EAAs) de novo, thus rely on dietary or symbiotic sources for them. Wood is a poor resource of nitrogen in general, and EAAs in particular. In this study, we investigated whether gut microbiota of the Asian longhorned beetle, Anoplophora glabripennis (Motschulsky), a cerambycid that feeds in the heartwood of healthy host trees, serve as sources of EAAs to their host under different dietary conditions. δ 13C-stable isotope analyses revealed significant δ 13C-enrichment (3.4 ± 0.1%; mean ± SEM) across five EAAs in wood-fed larvae relative to their woody diet. δ 13C values for the consumers greater than 1% indicate significant contributions from non-dietary EAA sources (symbionts in this case). In contrast, δ 13Cenrichment of artificial diet-fed larvae (controls) relative to their food source was markedly less (1.7 ± 0.1%) than was observed in wood-fed larvae, yet still exceeded the threshold of 1%. A predictive model based on δ 13Ceaa signatures of five EAAs from representative bacterial, fungal, and plant samples identified symbiotic bacteria and fungi as the likely supplementary sources of EAA in wood-fed larvae. Using the same model, but with an artificial diet as the dietary source, we identified minor supplementary bacterial sources of EAA in artificial diet-fed larvae. This study highlights how microbes associated with A. glabripennis can serve as a source of EAAs when fed on nutrient-limited diets, potentially circumventing the dietary limitations of feeding on woody substrates.

Host plant species determines symbiotic bacterial community mediating suppression of plant defenses

Herbivore associated bacteria are vital mediators of plant and insect interactions. Host plants play an important role in shaping the gut bacterial community of insects. Colorado potato beetles (CPB; Leptinotarsa decemlineata) use several Solanum plants as hosts in their natural environment. We previously showed that symbiotic gut bacteria from CPB larvae suppressed jasmonate (JA)-induced defenses in tomato. However, little is known about how changes in the bacterial community may be involved in the manipulation of induced defenses in wild and cultivated Solanum plants of CPB. Here, we examined suppression of JA-mediated defense in wild and cultivated hosts of CPB by chemical elicitors and their symbiotic bacteria. Furthermore, we investigated associations between the gut bacterial community and suppression of plant defenses using 16 S rRNA amplicon sequencing. Symbiotic bacteria decreased plant defenses in all Solanum hosts and there were different gut bacterial communities in CPB fed on different host plants. When larvae were reared on different hosts, defense suppression differed among host plants. These results demonstrate that host plants influence herbivore gut bacterial communities and consequently affect the herbivore’s ability to manipulate JA-mediated plant defenses. Thus, the presence of symbiotic bacteria that suppress plant defenses might help CPB adapt to host plants.

Improved Efficiency for Quantitative and Qualitative Indexing for Citrus tristeza virus and Citrus psorosis virus

Reverse transcription-polymerase chain reaction (RT-PCR) assays were developed for the detection of Citrus tristeza virus (CTV; genus Closterovirus) and Citrus psorosis virus (CPsV; genus Ophiovirus) in citrus trees. Real-time TaqMan RT-PCR was also developed for the detection of CTV. Three different sample preparation methods were compared. The total RNA extraction method by Qiagen was found to be more reliable than the other two methods consisting of crude plant sap extraction and total nucleic acid trapping on a silica bed. Of 287 samples tested for CTV, 210 samples tested positive by RT-PCR and 198 samples by enzyme-linked immunosorbent assay (ELISA). Furthermore, the results from monthly tests of a selected number of field-grown CTV-infected trees showed that RT-PCR detected the virus in 100% of the infected trees in winter and summer, whereas ELISA did not. The one-tube RT-PCR detection was developed for CPsV and was more sensitive than ELISA. Notably, three of 10 CPsV isolates were not detected by ELISA. As demonstrated here, our approach allows the efficacious detection of different viruses in citrus plants using a minimal amount of tissue during all seasons. The molecular methods described could be used in citrus certification programs and to test trees in nurseries and commercial orchards.

Colorado potato beetle manipulates plant defenses in local and systemic leaves

Herbivore microbial associates can affect diverse interactions between plants and insect herbivores. Some insect symbionts enable herbivores to expand host plant range or to facilitate host plant use by modifying plant physiology. However, little attention has been paid to the role of herbivore-associated microbes in manipulating plant defenses. We have recently shown that Colorado potato beetle secrete the symbiotic bacteria to suppress plant defenses. The bacteria in oral secretions from the beetle hijack defense signaling pathways of host plants and the suppression of induced plant defenses benefits the beetle’s performance. While the defense suppression by the beetle-associated bacteria has been investigated in local damaged leaves, little is known about the effects of the symbiotic bacteria on the manipulation of plant defenses in systemic undamaged leaves. Here, we demonstrate that the symbiotic bacteria suppress plant defenses in both local and systemic tissues when plants are attacked by antibiotic-untreated larvae.