XatA, an AT-1 autotransporter important for the virulence of Xylella fastidiosa Temecula1.

Xylella fastidiosa Temecula1 is the causative agent of Pierce's disease of grapevine, which is spread by xylem-feeding insects. An important feature of the infection cycle is the ability of X. fastidiosa to colonize and interact with two distinct environments, the xylem of susceptible plants and the insect foregut. Here, we describe our characterization of XatA, the X. fastidiosa autotransporter protein encoded by PD0528. XatA, which is classified as an AT-1 (classical) autotransporter, has a C-terminal β-barrel domain and a passenger domain composed of six tandem repeats of approximately 50 amino acids. Localization studies indicate that XatA is present in both the outer membrane and membrane vesicles and its passenger domain can be found in the supernatant. Moreover, XatA is important for X. fastidiosa autoaggregation and biofilm formation based on mutational analysis and the discovery that Escherichia coli expressing XatA acquire these traits. The xatA mutant also shows a significant decrease in Pierce's disease symptoms when inoculated into grapevines. Finally, X. fastidiosa homologs to XatA, which can be divided into three distinct groups based on synteny, form a single, well-supported clade, suggesting that they arose from a common ancestor

Getting rid of gut microbes: surface sterilization cleans symbionts from the insects'egg masses.

Diverse heteropteran insects that feed on economic important crops, commonly known as stink bugs, are associated with specific gut symbiotic bacteria within their midgut cryptic spaces or the gastric caeca. Recent studies have revealed that the stink bugs Nezara viridula, Acrosternum hilare, Murgantia histrionica, Euschistus heros, Chlorochroa ligata, Chlorochroa sayi, Chlorochroa uhleri, Plautia stali, Thyanta pallidovirens, Dichelops melacanthus, Edessa meditabunda, Loxa deducta, Pellaea stictica, Piezodorus guildinii, Thyanta perditor, (all within Pentatomidae family) carried one major bacterium in their midgut. Phylogenetic tree generated using the 16S rRNA gene sequences obtained from the midgut of these previous species placed all symbionts in a clade with the plant-associated bacteria Erwinia and Pantoea species. In this system, females of stink bug vertically transmit the symbionts by smearing them on the surface of the egg masses. When first instars of nymphs hatch, they probe the egg surface and orally acquire the symbionts. Once ingested, these microbial symbionts will reach the 4th section of the ventriculum (V4), also known as gastric caecum, where they establish. It was hypothesized that nymphs born from surface sterilized eggs do not carry the caeca-associated symbionts. Once ingested these microbial symbionts will reach the 4th section of the ventriculum (V4), also known as gastric caecum, where they establish. It was hypothesized that nymphs born from surface sterilized eggs do not carry the caeca-associated symbionts. Herne, using scanning eletron microscopy (SEM), we are showing that surface sterilized eggs do not carry the surface microbes while eggs from the field of E. heros, D. melacanthus, N. viridula, P. stitica, and Pi. Guildinii carry them. In this unique model of transmission where the symbionts located temporally on the surface of eggs, environment factors may have a great impact on bacterial survival. Additionally, climate change may impact the insect host ecology. This information may lead to design new strategies to control the stink bugs that could used in the integrated pest management

Phenolics, flavonoids and antioxidant capacities in Citrus species with different degree of tolerance to Huanglongbing

Huanglongbing (HLB) is a highly destructive disease to the citrus industry in Florida caused by the bacterium, Candidatus Liberibacter asiaticus(CLas) and is transmitted by Diaphorina citri. It is hypothesized that plants with high phenolic contents show higher tolerance to certain plant pathogens. In this regard, different citrus varieties and plants of genera related to Citrus were analyzed for their total phenolic and flavonoids contents, and their antioxidant capacities. In addition, the secondary metabolites in the leaves of seven citrus species were analyzed using high performance liquid chromatographymass spectrometry (HPLC-MS). Colorimetric assays showed that curry leaf contained the highest total phenolic content and free radical scavenging activity (DPPH). Curry leaf also contained high concentrations of an unusual class of carbazole alkaloids. Tolerant Citrus species contained high levels of phenolics and flavonoids and showed high antioxidant capacities. Our results suggest that high phenolic and flavonoid leaf contents correlate with increased citrus tolerance to CLas bacterium. The results also suggest that the high level of carbazole alkaloids, known for their strong antimicrobial properties in curry leaf, could make it immune to the CLas bacteria. Understanding the mechanisms underpinning citrus tolerance to HLB will contribute to the development of commercially tolerant citrus cultivars.Acknowledgments We thank Yasser Nehela for the technical assistance and Lorraine Jones for maintaining the trees in greenhouses. This work was kindly funded by Citrus Research and Development Foundation, grant number 19-015. Funding This work was supported by the Citrus Research and Development Foundation [19-015]

Translocation of Oxytetracycline in Citrus Plants after Root Drench and Stem Delivery

Huanglongbing (HLB) is the most destructive disease to the citrus industry. In North America, HLB is caused by Candidatus Liberibacter asiaticus (CLas) and is transmitted by the Asian citrus psyllid, Diaphorina citri. Recent studies showed that antibiotics such as oxytetracycline and streptomycin were effective against the CLas pathogen in planta. The objectives of this study were to investigate the uptake, translocation, and stability of oxytetracycline in citrus seedlings. Oxytetracycline was delivered via root or stem. The level of oxytetracycline in treated plants was monitored using high-performance liquid chromatography (HPLC) and/or enzyme-linked immunosorbent assay (ELISA). The HPLC and the ELISA methods showed similar results at high concentrations; however, the ELISA was more sensitive than the HPLC method. The highest level of oxytetracycline after root incubation was found in roots, followed by stem-xylem, stem-phloem, and in leaves. On the other hand, the level of oxytetracycline in the xylem and phloem was higher than that found in the root when delivered via stem. Oxytetracycline was still detectable in all tested tissues thirty-five days after treatment, indicating that oxytetracycline was relatively stable in citrus plants and could inhibit CLas growth for a few months in the field

Uptake, Translocation, and Stability of Oxytetracycline and Streptomycin in Citrus Plants

Huanglongbing (HLB), or citrus greening, is the most destructive disease to the citrus industry. In Florida, it is caused by the bacterium, Candidatus Liberibacter asiaticus (CLas) and is transmitted by the Asian citrus psyllid, Diaphorina citri. Recent studies suggested that antibiotics could inhibit the growth of the CLas pathogen in planta. In the current study, we investigated the uptake and translocation of oxytetracycline and streptomycin in citrus seedlings. Oxytetracycline and streptomycin were delivered via root and stem and their level in various tissues was monitored using enzyme-linked immunosorbent assay (ELISA). Oxytetracycline and streptomycin were detected in the leaves, xylem, phloem, and root after root drench and stem delivery. High levels of antibiotics were detected in the roots after root drench, whereas high levels of antibiotics were detected in the canopy after stem delivery. The level of oxytetracycline detected in the phloem, xylem, and leaves after root drench was higher than that of streptomycin. Whereas the level of streptomycin in root was higher than that of oxytetracycline, indicating that streptomycin was bound to the xylem tissues. Oxytetracycline and streptomycin were detected in the phloem, xylem, leaves, and root tissues thirty-five days after the root incubation in 200 μg.ml-1 solution. These results demonstrated that oxytetracycline and streptomycin were relatively stable and could inhibit CLas growth for a couple of months in citrus trees. Observations reported in this study regarding the distribution and stability of oxytetracycline and streptomycin in citrus plants could be useful for designing an effective program for the control of HLB disease using antibiotics.Funding: National Institute of Food and Agriculture: 2019-70016-29096 Acknowledgments: We thank our lab members for the helpful discussion and technical assistance. This work was generously funded by a grant # 2019-70016-29096 from NIFA-SCRI for OB and NK. Conflicts of Interest: Page: 10 The authors declare no conflict of interes

Tracing Penicillin Movement in Citrus Plants Using Fluorescence-Labeled Penicillin

Huánglóngbìng (HLB), citrus greening, is one of the most destructive diseases of citrusplants worldwide. In North America, HLB is caused by the phloem-limited bacteriumCandidatusLiberibacter asiaticus and is transmitted by the Asian citrus psyllid,Diaphorina citri. No cure exists atpresent, and the use of antibiotics for the control of HLB has gained interest due to the significantlosses to the citrus industry. Because of unsatisfactory results when using foliar applications ofantibiotics, concerns were raised regarding the uptake and translocation of these materials within trees.We, therefore, investigated a method that allows us to study the movement of antibiotic materialsin citrus plants. Herein, we utilized a fluorescence-labeled penicillin, BOCILLIN™FL-Penicillin(FL-penicillin), to study the uptake and translocation of penicillin in citrus plants. FL-penicillin wasapplied by puncture to the stem of young citrus seedlings and was traced by using fluorescencemicroscopy. After application, we detected FL-penicillin in the leaves and in the stem xylem andphloem tissues above and below the application site in both intact and partially bark-girdled citrusseedlings, indicating that it is easily taken up and transported through the plant vascular system.In addition, we detected FL-penicillin in the gut ofD. citri, which were allowed to feed on thetreated plants, suggesting translocation of this molecule into the vascular tissue. We propose thatthe use of fluorescent-labeled molecules could be an effective tool for understanding the uptake andtranslocation of antibiotics and other macromolecules in plants and insects.Funding:This research was funded by the National Institute of Food and Agriculture, grant number2019-70016-29096.Acknowledgments:We thank our lab members for helpful discussion and technical assistance. This work wasgenerously funded by the USDA NIFA-SCRI award number 2019-70016-29096

The abundant extrachromosomal DNA content of the Spiroplasma citri GII3-3X genome

<p>Abstract</p> <p>Background</p> <p><it>Spiroplama citri</it>, the causal agent of citrus stubborn disease, is a bacterium of the class <it>Mollicutes </it>and is transmitted by phloem-feeding leafhopper vectors. In order to characterize candidate genes potentially involved in spiroplasma transmission and pathogenicity, the genome of <it>S. citri </it>strain GII3-3X is currently being deciphered.</p> <p>Results</p> <p>Assembling 20,000 sequencing reads generated seven circular contigs, none of which fit the 1.8 Mb chromosome map or carried chromosomal markers. These contigs correspond to seven plasmids: pSci1 to pSci6, with sizes ranging from 12.9 to 35.3 kbp and pSciA of 7.8 kbp. Plasmids pSci were detected as multiple copies in strain GII3-3X. Plasmid copy numbers of pSci1-6, as deduced from sequencing coverage, were estimated at 10 to 14 copies per spiroplasma cell, representing 1.6 Mb of extrachromosomal DNA. Genes encoding proteins of the TrsE-TraE, Mob, TraD-TraG, and Soj-ParA protein families were predicted in most of the pSci sequences, in addition to members of 14 protein families of unknown function. Plasmid pSci6 encodes protein P32, a marker of insect transmissibility. Plasmids pSci1-5 code for eight different <it>S. citri </it>adhesion-related proteins (ScARPs) that are homologous to the previously described protein P89 and the <it>S. kunkelii </it>SkARP1. Conserved signal peptides and C-terminal transmembrane alpha helices were predicted in all ScARPs. The predicted surface-exposed N-terminal region possesses the following elements: (i) 6 to 8 repeats of 39 to 42 amino acids each (sarpin repeats), (ii) a central conserved region of 330 amino acids followed by (iii) a more variable domain of about 110 amino acids. The C-terminus, predicted to be cytoplasmic, consists of a 27 amino acid stretch enriched in arginine and lysine (KR) and an optional 23 amino acid stretch enriched in lysine, aspartate and glutamate (KDE). Plasmids pSci mainly present a linear increase of cumulative GC skew except in regions presenting conserved hairpin structures.</p> <p>Conclusion</p> <p>The genome of <it>S. citri </it>GII3-3X is characterized by abundant extrachromosomal elements. The pSci plasmids could not only be vertically inherited but also horizontally transmitted, as they encode proteins usually involved in DNA element partitioning and cell to cell DNA transfer. Because plasmids pSci1-5 encode surface proteins of the ScARP family and pSci6 was recently shown to confer insect transmissibility, diversity and abundance of <it>S. citri </it>plasmids may essentially aid the rapid adaptation of <it>S. citri </it>to more efficient transmission by different insect vectors and to various plant hosts.</p

The Major Antigenic Membrane Protein of “Candidatus Phytoplasma asteris” Selectively Interacts with ATP Synthase and Actin of Leafhopper Vectors

Phytoplasmas, uncultivable phloem-limited phytopathogenic wall-less bacteria, represent a major threat to agriculture worldwide. They are transmitted in a persistent, propagative manner by phloem-sucking Hemipteran insects. Phytoplasma membrane proteins are in direct contact with hosts and are presumably involved in determining vector specificity. Such a role has been proposed for phytoplasma transmembrane proteins encoded by circular extrachromosomal elements, at least one of which is a plasmid. Little is known about the interactions between major phytoplasma antigenic membrane protein (Amp) and insect vector proteins. The aims of our work were to identify vector proteins interacting with Amp and to investigate their role in transmission specificity. In controlled transmission experiments, four Hemipteran species were identified as vectors of “Candidatus Phytoplasma asteris”, the chrysanthemum yellows phytoplasmas (CYP) strain, and three others as non-vectors. Interactions between a labelled (recombinant) CYP Amp and insect proteins were analysed by far Western blots and affinity chromatography. Amp interacted specifically with a few proteins from vector species only. Among Amp-binding vector proteins, actin and both the α and β subunits of ATP synthase were identified by mass spectrometry and Western blots. Immunofluorescence confocal microscopy and Western blots of plasma membrane and mitochondrial fractions confirmed the localisation of ATP synthase, generally known as a mitochondrial protein, in plasma membranes of midgut and salivary gland cells in the vector Euscelidius variegatus. The vector-specific interaction between phytoplasma Amp and insect ATP synthase is demonstrated for the first time, and this work also supports the hypothesis that host actin is involved in the internalization and intracellular motility of phytoplasmas within their vectors. Phytoplasma Amp is hypothesized to play a crucial role in insect transmission specificity

Ralstonia syzygii, the Blood Disease Bacterium and Some Asian R. solanacearum Strains Form a Single Genomic Species Despite Divergent Lifestyles

The Ralstonia solanacearum species complex includes R. solanacearum, R. syzygii, and the Blood Disease Bacterium (BDB). All colonize plant xylem vessels and cause wilt diseases, but with significant biological differences. R. solanacearum is a soilborne bacterium that infects the roots of a broad range of plants. R. syzygii causes Sumatra disease of clove trees and is actively transmitted by cercopoid insects. BDB is also pathogenic to a single host, banana, and is transmitted by pollinating insects. Sequencing and DNA-DNA hybridization studies indicated that despite their phenotypic differences, these three plant pathogens are actually very closely related, falling into the Phylotype IV subgroup of the R. solanacearum species complex. To better understand the relationships among these bacteria, we sequenced and annotated the genomes of R. syzygii strain R24 and BDB strain R229. These genomes were compared to strain PSI07, a closely related Phylotype IV tomato isolate of R. solanacearum, and to five additional R. solanacearum genomes. Whole-genome comparisons confirmed previous phylogenetic results: the three phylotype IV strains share more and larger syntenic regions with each other than with other R. solanacearum strains. Furthermore, the genetic distances between strains, assessed by an in-silico equivalent of DNA-DNA hybridization, unambiguously showed that phylotype IV strains of BDB, R. syzygii and R. solanacearum form one genomic species. Based on these comprehensive data we propose a revision of the taxonomy of the R. solanacearum species complex. The BDB and R. syzygii genomes encoded no obvious unique metabolic capacities and contained no evidence of horizontal gene transfer from bacteria occupying similar niches. Genes specific to R. syzygii and BDB were almost all of unknown function or extrachromosomal origin. Thus, the pathogenic life-styles of these organisms are more probably due to ecological adaptation and genomic convergence during vertical evolution than to the acquisition of DNA by horizontal transfer