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Huanglongbing (HLB) is a deadly, incurable citrus disease putatively caused by the unculturable bacterium, 'Candidatus Liberibacter asiaticus' (CLas), and transmitted by Diaphorina citri. Prior studies suggest D. citri transmits CLas in a circulative and propagative manner; however, the precise interactions necessary for CLas transmission remain unknown, and the impact of insect sex on D. citri-CLas interactions is poorly understood despite reports of sex-dependent susceptibilities to CLas. We analyzed the transcriptome, proteome, metabolome, and microbiome of male and female adult D. citri reared on healthy or CLas-infected Citrus medica to determine shared and sex-specific responses of D. citri and its endosymbionts to CLas exposure. More sex-specific than shared D. citri responses to CLas were observed, despite there being no difference between males and females in CLas density or relative abundance. CLas exposure altered the abundance of proteins involved in immunity and cellular and oxidative stress in a sex-dependent manner. CLas exposure impacted cuticular proteins and enzymes involved in chitin degradation, as well as energy metabolism and abundance of the endosymbiont 'Candidatus Profftella armatura' in both sexes similarly. Notably, diaphorin, a toxic Profftella-derived metabolite, was more abundant in both sexes with CLas exposure. The responses reported here resulted from a combination of CLas colonization of D. citri as well as the effect of CLas infection on C. medica. Elucidating these impacts on D. citri and their endosymbionts contributes to our understanding of the HLB pathosystem and identifies the responses potentially critical to limiting or promoting CLas acquisition and propagation in both sexes.

The Asian citrus psyllid (Diaphorina citri Kuwayama) is an invasive Homopteran that has crippled citrus production in Florida with the spread of the Huanglongbing (Citrus Greening) disease, which yields small discolored and bitter fruit. The disease is associated with the bacterium ‘Candidatus’ Liberibacter and is rapidly spreading to other citrus producing states. Gene targets were competitively deposited by experts from diverse fields for RNAi screening through an Innocentive challenge funded by the Citrus Research and Development Foundation. Four of the fifty genes were identified in dsRNA feeding trials to induce both significantly high mortality as well as concentration dependent dose response. Three of these are involved in redox biochemistry. These results indicate that gene silencing of proteins involved in metabolism by redox chemistry can induce mortality at low doses and that functional targeting in this organism may be the best strategy for psyllid control by RNAi.

Abstract Background Developing monocots that accumulate more vegetative tissue protein is one strategy for improving nitrogen-sequestration and nutritive value of forage and silage crops. In soybeans (a dicotyledonous legume), the vspA and B genes encode subunits of a dimeric vegetative storage protein that plays an important role in nitrogen storage in vegetative tissues. Similar genes are found in monocots; however, they do not accumulate in leaves as storage proteins, and the ability of monocot leaves to support accumulation of an ectopically expressed soybean VSP is in question. To test this, transgenic maize (Zea Mays L. Hi-II hybrid) lines were created expressing soybean vspB from a maize ubiquitin Ubi-1 promoter. Results From 81 bombardments, 101 plants were regenerated, and plants from five independent lines produced vspB transcripts and VSPβ polypeptides. In leaves from seven-week-old plants (prior to flowering), VSPβ accumulated to 0.5% of the soluble leaf protein in primary transgenic plants (R0), but to only 0.03% in R1 plants. During seed-filling (silage-stage) in R1 plants, the VSPβ protein was no longer detected in leaves and stems despite continued presence of the vspB RNA. The RNA transcripts for this peptide either became less efficiently translated, or the VSPβ protein became unstable during seed-fill. Conclusion Developmental differences in the accumulation of soybean VSPβ when transgenically expressed in maize show that despite no changes in the vspB transcript level, VSPβ protein that is readily detected in leaves of preflowering plants, becomes undetectable as seeds begin to develop.

Huanglongbing (HLB), referred to as citrus greening disease, is a bacterial disease impacting citrus production worldwide and is fatal to young trees and mature trees of certain varieties. In some areas, the disease is devastating the citrus industry. A successful solution to HLB will be measured in economics: citrus growers need treatments that improve tree health, fruit production, and most importantly, economic yield. The profitability of citrus groves is the ultimate metric that truly matters when searching for solutions to HLB. Scientific approaches used in the laboratory, greenhouse, or field trials are critical to the discovery of those solutions and to estimate the likelihood of success of a treatment aimed at commercialization. Researchers and the citrus industry use a number of proxy evaluations of potential HLB solutions; understanding the strengths and limitations of each assay, as well as how best to compare different assays, is critical for decision-making to advance therapies into field trials and commercialization. This perspective aims to help the reader compare and understand the limitations of different proxy evaluation systems based on the treatment and evaluation under consideration. The researcher must determine the suitability of one or more of these metrics to identify treatments and predict the usefulness of these treatments in having an eventual impact on citrus production and HLB mitigation. As therapies advance to field trials in the next few years, a reevaluation of these metrics will be useful to guide future research efforts on strategies to mitigate HLB and vascular bacterial pathogens in other perennial crops., Competing Interests: The author(s) declare no conflict of interest.

Background: Citrus Huanglongbing (HLB) is a bacterial disease with high economic significance. The associated agent Candidatus Liberibacter asiaticus is a fastidious, phloem-limited, intracellular bacterium that is transmitted by an insect vector the Asian citrus psyllid (ACP). The genome of Ca. L. asiaticus contains protein secretion machinery that suggests host cell modulation capacity of this bacterium., Results: A total of 28 candidate effectors, an important class of secreted proteins, were predicted from the Ca. L. asiaticus genome. Sequence specific primers were designed for reverse transcription (RT) and quantitative PCR (qPCR), and expression was validated for 20 of the effector candidates in infected citrus with multiple genetic background. Using detached leaf inoculation, the mRNA of effectors was detected from 6 h to 7 days post ACP exposure. It was observed that higher bacterial titers were associated with a larger number of effectors showing amplification across all samples. The effectors' expression were compared in citrus hosts with various levels of HLB tolerance, including susceptible Duncan grapefruit and Washington navel orange, tolerant citron and Cleopatra mandarin, and resistant Pomeroy trifoliate and Carrizo citrange. Across all genotypes relatively high expression was observed for CLIBASIA_03695, CLIBASIA_00460, CLIBASIA_00420, CLIBASIA_04580, CLIBASIA_05320, CLIBASIA_04425, CLIBASIA_00525 and CLIBASIA_05315 in either a host-specific or -nonspecific manners. The two genotypes in each HLB-response group also show effector-expression profiles that seem to be different. In a companion study, the expression of effectors was compared between leaves and roots of own-rooted citrus that had been Ca. L. asiaticus-infected for more than a year. Results indicated relatively high expression of CLIBASIA_03875, CLIBASIA_04800 and CLIBASIA_05640 in all leaf and some root tissues of citron, Duncan and Cleopatra., Conclusion: This temporal and spatial expression analysis of Ca. L. asiaticus effectors identified candidates possibly critical for early bacterial colonization, host tolerance suppression and long-term survival which are all worthy of further investigation.

Background: Salivary sheaths, also known as stylet sheaths or stylet tracks, are essential features of the piercing-sucking feeding mechanism of plant-feeding hemipteran insects, many of which are vectors of economically important plant viral and bacterial pathogens. Although knowledge of their structure and function is incomplete, these salivary sheaths are frequently used by researchers to study hemipteran's feeding behavior, host preference, or host resistance, because these sheaths remain in the plant tissues after the insect withdraws its stylets following its feeding or probing on these tissues. However, in most cases, it is not known how long these salivary sheaths may last in plant tissues after their deposition by the feeding insects. An earlier report suggested that the salivary sheaths of the Asian citrus psyllid, Diaphorina citri (Hemiptera, Liviidae), vector of the devastating huanglongbing (citrus greening) disease bacterium, start to dissipate 1 week after their deposition in citrus leaves., Methods and Results: Here, using epifluorescence microscopy of cross sections in citron leaves, we found that D. citri salivary sheaths show signs of degradation in 3-4 weeks and become mostly degraded by 5-6 weeks, following their deposition by the psyllids into citrus tissues. Degradation of the salivary sheath starts at or near the "flange" area close to the leaf surface and continues gradually inward through the intercellular part of the sheath, within the mesophyll tissue, but apparently does not extend to the deeper or intracellular parts of the sheath in or near the phloem. Staining citron leaf sections with the fluorescent stain calcofluor white, which stains fungi, or propidium iodide (DNA/RNA stain) suggested that the degraded parts of the older salivary sheaths are not associated with fungi or bacterial accumulations., Conclusion: We speculate that degradation of the salivary sheaths may be due to enzymatic activities in the host plant, especially in the extracellular matrix of the mesophyll tissue., Competing Interests: There are no conflicts of interest.