MtCOI haplotyping. Additional samples are gradually received for mtCOI haplotyping. A total of 250 samples were analyzed, including from a center of diversity in Asia, and elsewhere are to explore baseline diversities within and between different locations, in an attempt to relate populations from the US and elsewhere in the Western Hemisphere to those from a primary region of endemism. Sequences (~1300 bases) are remarkably similar, suggesting the need for a different marker. This result was surprising, considering the widespread use of the COI for homopteran bar-coding. Time-course and transmission studies were established to assess transmission frequency in relation to qPCR and/or dot blot hybridization detection of Ca. Liberibacter solanacearum (AZ) and in Ca. L. asiaticus (FL) in individual psyllids reared on infected plant material. Single potato psyllids reared on infected tomato and given a range of inoculation access feeds on tomato from 30 min, and 1,2, 4hr-transmitted Ca. L. solanacearum 20, 35, 30, and 75% of the time (20 plants per rep). When five psyllids were used transmission frequency was 5, 35, 25, and 70%, respectively. Studies suggest that once acquired psyllids transmit at relatively high frequency (70%); tests are underway for potato psyllid at 8,12,and 24 hrs. The goal is to select individuals for light microscopy, and SEM-TEM ultrastructural observations to understand the dynamic relationship over a range of different AAP and IAPs. IAP studies are underway for Ca. Lsol using tomato seedlings for bioassay, together with PCR or qPCR monitoring, all difficult undertakings owing to the apparent uneven distribution of bacteria in plants, which confounds reliable detection for localization studies. Transmission and scanning electron microscope studies focused on large monocultures [interpreted as thick biofilms] of rod-shaped, fastidious bacteria that are consistently associated with the alimentary canal, from the oral box (including salivary glands) to the posterior midgut, of infected but not uninfected psyllids, and most consistently observed in 3rd instar and older psyllids. In addition, studies have revealed the dynamic behavior in the positions that the intestine assumes within the abdomen of live psyllids. As with the causal, bacterial agent for Pierce’s disease, attempts to reliably implement FISH for determining the time-course proliferation and gross anatomical affinities of the target pathogen within the psyllid vector body remain tentative. We are implementing colloidal gold – DNA hybridization for ultrastructural studies. 100nm thick, plastic Z-section libraries of the oral box have been made from infected and non-infected potato psyllids to map the tissue/organ organization where bacteria reside. The net accomplishments are visualization of Ca. Liberibacter in the newly resolved anatomical structures, and constructing models for mode and pathway of transmission. In research designed to prove Koch`s postulates for CLso, we determined that infected plant symptoms do not correlate with signal detection in qPCR. We have developed a culture technique that sustains the bacterium for a period of two weeks in liquid medium. We were able to show that clean Psyllids that were never exposed to CLso when fed on artificial feeding solution containing infected psyllid gut extracts are able to acquire CLso detectable by regular PCR and qPCR for OMP gene of CLso. Using TEM, we were able to determine average length of this bacterium to be 3.52um and average diameter to be 0.15um. qPCR on near pure cultures show bacteria survive and reproduce. Unusual and polymorphic shapes of CLso cells observed in and on internal psyllid organs suggest that they do not form a typical septum as is common during binary fission of many bacteria. Two-three manuscripts reporting these results will be submitted.
In this continuation we are (i) utilizing mined EST data in PAVE to validate bioinformatically predicted effectors using RNAi knock down analyses (feeding, microinjection), together with FISH localization of mRNA, and qPCR quantification; additional putative protein effectors will be identified in the secretome (proteome of dissected salivary glands and guts; candidate EST-expressed proteins), (ii) initiating efforts to implement yeast-2 hybrid to elucidate protein-protein interactors in ACP adults and Las expression libraries (psyllids courtesy K. Stelinski), and (iii) validation by in vivo pull down assays. Classes of targets of interest will be based on comparative SEM-TEM evidence together with transcriptomic- and bioinformatics-driven (EST) predictions thus far include the psyllid secretome, vector innate immunity and RNA silencing pathways, nutrition-metabolism, and Las adhesion and virulence-pathogenicity factors predicted based on the Las genome sequence. Purative effector proteins will thereby be identified using a multi-pronged, progressive approach. The goal is to implement new knowledge of the key effectors identified here to inform genetics based approaches for abating vital secretome functions required for psyllid livelihood and nourishment, (ii) Las infection and circulation, and (ii) transmission to control citrus greening disease. Comparative analyses of ACP and PP psyllid transcriptomes revealed that ~60% of the transcripts were common to both species suggestive of roles in core growth and developmental processes which are ideal targets for RNA interference (RNAi) with the goal to interfere with psyllid survivability. These kinds of target are also good for initial optimization of RNAi using mortality bioassays. A number of these genes were selected and experimentally validated by RT-PCR in ACP adults. Additionally, in silico expressional analyses of ACP and PP transcripts in response to Liberibacter infection have been conducted, resulting in the identification of a large number of up- and down-regulated transcripts (>2 fold) in infected psyllid nymphs and adults. BLAST (NCBI) searches and gene ontology gene predictions indicate these genes are likely involved in key psyllid-Liberibacter interactions such as in virulence, adhesion, and immunity, which could be lucrative targets for RNAi to interfere with survival, propagation, circulation, pathogenicity, and ultimately transmission of Ca. Liberibacter. RNAi assays are under development employing a system developed previously for whitefly artificial feeding to orally deliver dsRNA corresponding to gut targets. The dsRNAs for selected targets are synthesized using in vitro transcription and the MEGAscript RNAi kit. Currently adult psyllids survive up to 5 days in this system. We also developed a quantitative qPCR detection method using the psyllid COI gene for normalization. Information about the molecular basis of key psyllid-Liberibacter interactions is lacking with respect to entry, metabolic pathways use, and immune system effectors and interactions with bacterial proteins. Proteins from guts and salivary glands of infected and noninfected potato psyllid adults were separated by SDS-PAGE (10’14% acrylamide). Each gel lane was digested with trypsin and peptides were analyzed by nanoLC-LTQ-Orbitrap (Thermoelectron) mass spectrometry. We identified 795 proteins in the gut. Of those, 212 proteins showed >1.5 fold increase in expression in infected compared to noninfected guts. Among these are a number of putative effectors with direct implication in bacterial transport and movement in the circulative, propagative pathway that have been selected for mRNA validation and qPCR analysis of expression levels in infected and uninfected adult ACP and PoP psyllids.
Expression in citrus of dsRNA targeting a psyllid gene, through use of the paratransgenic CTV expression vector, was further characterized. Our analysis showed that mortality of psyllids feeding on citrus producing target dsRNAs was directly correlated with accumulation of total psyllid gene RNA (ssRNA + dsRNA) produced within the leaf tissue. As much as 80 to 90% mortality of adult psyllids was observed after 6 days of feeding on leaves with the highest level of psyllid target gene RNA. Citrus leaves expressing RNA from the green fluorescent protein (GFP) cloned into the CTV expression vector induced no mortality in adult psyllids. These results support the hypothesis that mortality is associated with psyllid gene specific dsRNA ingestion. Currently experiments are being conducted on performance of all psyllid life cycle stages.
Progress with the rapid flowering system (pvc pipe scaffolding system) in the greenhouse: Selected transgenic plants produced from juvenile explant, budded to precocious tetraploid rootstocks in airpots are growing well in our RES system, with some plants reaching 8 feet in height. Additional transgenics were propagated onto additional new rootstocks expected to reduce juvenility, including the somatic hybrid Amblycarpa + Flying Dragon. The goal is to reduce juvenility by several years to accelerate flowering and fruiting of the transgenic plants. Experiments to efficiently stack promising transgenes are underway. The first transformation experiments using the two-transgene Gateway based cloned construct combining our best transgene for HLB resistance (NPR-1 from Arabidopsis) with our best transgene against canker that also has some affect on HLB (the synthetic CEME lytic peptide gene) were initiated, and so far 30 putative transgenic lines of the sweet orange cultivars Hamlin and Valencia have been regenerated. These plantlets have been micrografted to Carrizo rootstock. The goal is to provide stable resistance to both HLB and canker, with transgene backup to prevent Liberibacter from overcoming single transgene resistance. Correlating transgene expression with disease resistance response: We continued work to optimize an ELISA protocol to detect lytic peptide in transgenic Citrus plants using the LIMA antibody. This protocol has should be useful for evaluating transgenic plants containing either LIMA or CEME antimicrobials, using the same antibody. Since most of our constructs have the C-myc tag, ELISA and Western blot protocols have been optimized for large scale rapid screening of the transgenic plants to identify those with maximum transgene expression. Improved transformation methodology (for seedless or recalcitrant cultivars, and eventually marker-free consumer-friendly transformation): A vector containing a dual T-DNA border has been constructed. To test the vector functionality and determine T-DNA segregation, we have incorporated a visual Anthocyanin expressing gene from Grape (VVMYB) into one of the T-DNA. This gene on expression turns cells purple. The other T-DNA contains a fusion negative-positive selectable marker gene for selection (codA/nptII; Vector 1). We are currently constructing another fusion negative-positive selectable marker gene, by replacing the nptII gene with a gene that encodes for resistance to the antibiotic hygromycin (hptII). This construct will be used for transformation of citrus cell suspension cultures (Vector 2).
Dr. Cecilia Zapata, PI, resigned August 2012. Before leaving, we planned for reduced effort until a replacement could be found. Dr. Vladimir Orbovic agreed to assist with oversight of the mature tissue transformation facility in the interim. Below is the quarterly report Dr. Orbovic submitted. In the first three months of the funding period, Mature Tissue Transformation Laboratory (MTTL) has undergone big changes. The person who supervised the Lab for the last three years has left that post in the beginning of September. In the anticipation of prolonged period without managerial supervision for MTTL, departing supervisor made a decision to discard high percentage of plants from the growth room to prevent accumulation of unused plants. The transformation experiments were scheduled at the rate of one per month. However, temporary supervisor revised the plan up to two experiments per month. To accommodate such change, certain batches of plants that were used a source of explants only once were not discarded as planned. Also, some of the smaller rootstock plants left for practice and as surplus were transplanted and will serve as an additional batch of rootstock plants. Throughout this period, nine co-incubation experiments were performed. Four of those experiments were done with Hamlin explants, four with Valencia explants, and one with Pineapple sweet orange explants. In the Hamlin experiments, 2390 explants were cut for treatment with Agrobacterium; 2520 explants were used in Valencia experiments, and 690 explants were used in Pineapple experiment. Here are the results of GUS assays: 270 shoots harvested from different experiments with Hamlin were tested and two were positive. Out of 210 tested shoots of Valencia harvested from different experiments, five were positive. And finally, out of 68 shoots of Pineapple orange harvested from two experiments, two were positive. One of two positive Valencia shoots died upon grafting. Other positive shoots appear healthy and will be moved to growth room soon. These results mark a milestone as all three commercially important cultivars of sweet orange were successfully transformed. There are four Ray Ruby plants completely cleaned from microorganisms and ready to become source of shoots for production of branches. These Ray Ruby plants were obtained from USDA as ‘clean’ although additional testing in MTTL has shown that they did harbor some microorganisms. Repeated micro-grafting of meristem regions to new and clean rootstock plants resulted in selection of plants that were purged of any pests.
The antibody developer, Creative Biolabs, Inc., identified six monoclonal antibodies to the 30 amino acid peptide antigen used, which corresponds to an extracellular loop of the Candidatus Liberibacter asiaticus outer membrane protein NodT. The materials were received by Dr. McNellis’ lab at Penn State University in September of 2012. Four of the antibodies appear to be useful for the project, based on molecular analyses of their binding efficiency to the epitope target and their structural integrity. This type of single-chain engineered monoclonal antibody is provided to us as a DNA clone, from which we express the antibody in bacteria. We are currently working on producing the antibodies in E. coli bacteria. This material will allow us to test whether the antibodies can be used to detect NodT protein in protein extracts from psyllids and citrus trees. The transformation construct for expressing the FLT-antiNodT fusion protein in citrus has been initiated and will be completed soon.
In recent seasons of freeze and drought episodes, symptomatic HLB-infected trees were much more affected by the extremes of temperature and moisture than trees without HLB. Symptoms of stress are excessive leaf loss and premature fruit drop by HLB-infected trees even when trees are managed with enhanced nutritional programs. This stress intolerance may indicate a lack of fibrous roots. To access root status of HLB affected trees, blocks of 2,307 three yr-old Hamlin orange and 2693 four yr-old Valencia orange trees were surveyed for PCR status and visual symptoms. The incidence of presymptomatic (PCR+, visually negative) and symptomatic (PCR+, visually positive) trees was 22 and 46% for the Hamlin block and 55 and 34% for the Valencia block, respectively. In a second survey, 10 to 25 yr-old Valencia trees were identified within 3-6 months of canopy expression as HLB symptomatic (HLB+, PCR+) or asymptomatic (HLB-, PCR-) in groves located in the central ridge, south-central and southwest flatwoods. Pairs of HLB+ and HLB- trees were evaluated for PCR status, fibrous roots and Phytophthora nicotianae progagules in rhizosphere soil. HLB+ trees had 33-49% less fibrous roots and higher P. nicotianae per root but populations were high on both HLB+ and HLB-. Impairment of nutrient and water uptake may result from the direct effect of HLB on root function and be exacerbated by the interaction with P. nicotianae on fibrous root loss.
Objective 1 (To define the role of chemotaxis in the location and early attachment to the leaf and fruit surface). Assays to determine the motility response of canker strains to different stimuli are in progress. Chemotaxis responses to stimuli for Xcc strains including types A, Aw and A* canker bacteria were confirmed to vary among strains. To determine the basis for these differences, an in silico study of genes involved in chemotaxis and adhesion was performed. This involves two approaches: 1) comparison of the methyl-accepting chemotaxis proteins (MCPs) for which complete sequence genome is available for Xanthomonads (X. citri subsp. citri type A, X. fuscans subsp. aurantifolia, X. alfalfae subsp. citrumelonis, X. campestris pv. campestris) and 2) amplification of fragments of MCPs and adhesins, using thirty specific PCR primer sets designed for these genes in Xanthomonads. Until now, minor differences among the different strains have been detected even between citrus and non-citrus bacteria. The MCPs of those strains with distinct gene sequences are currently being analyzed to verify the relationship between genetic variation and chemotaxis response to different stimuli. Objective 2 (To investigate bifofilm formation and composition and its relationship with bacteria structures related with motility in different strains of Xcc and comparison to non canker causing xanthomonads). Investigation is now focused on the study of bacterial appendages involved in formation of the biofilm matrix. Analysis by SDS-PAGE gel electrophoresis showed no qualitative differences among the different Xanthomonads. Because differences the morphology of appendages were seen by electron microscopy, potential quantitative modifications are under evaluation utilizing differential gene expression of genes involved in formation of the appendages. Specific primers and real time RT-PCR are being developed to quantify expression of type IV pilus and flagellar genes for bacteria in the swarming, planktonic and biofilm formation stages.
From the three FT constructs created (FT1, FT2, FT3), the FT3 construct has shown significant induction of flowering on transformed tobacco plants. FT3 shows promising results at shortening the juvenility period in both citrus and tobacco. Compared to a wildtype control of tobacco the flowering time of F2 generation plants transformed with the FT3 construct is over 3 months earlier. A multi-faceted approach to understand the activity of citrus FT is underway. One of the approaches involves measuring expression levels of FT1, FT2, and FT3 in different Citrus varieties using Real Time PCR to determine FT behavior at different stages of growth. Analysis for the first 3 months has been performed on Pummelo and pineapple sweet orange varieties and all three genes are actively transcribed at different levels depending on the time period. The biological effects of various phytohormones such as ethylene and gibberellic acid on FT3 expression and flowering will be monitored. This approach will allow us to devise a method to delay flowering induction at early stages in citrus due to the observed premature formation of flowers at tissue culture stages. The final approach is to isolate the FT3 mobile protein and introduce it into phloem of citrus and tobacco through various methods in order to induce early flowering. The protein will be synthesized and various trials of exogenous protein application will be performed. This approach will allows us to create a practical protocol for shortening juvenility periods. In both citrus and tobacco the FT3 genomic construct with the constitutive FMV promoter is highly effective, causing very early flowering. Unfortunately, in citrus, the flowering occurs on the plate, before the transformed material is useable. Some work has been done in an attempt to control the speed of flowering using day length, temperature, and gibberellic acid. In a further attempt to control the activity of the FT3 gene, a construct using an inducible promoter is being produced. This inducible promoter is based on the activity of an ecdysone receptor and is induced using the chemical methoxyfenozide. Before this construct is developed, the effectiveness of the FT3 cDNA is being compared to the FT3 genomic DNA using the original FMV promoter in the hopes that the smaller cDNA will be just as effective and can be used in the new construct without changing the flowering character.
During this period ‘Duncan’ grapefruit (considered susceptible to HLB) and ‘Sun Chu Sha’ mandarin (considered moderately tolerant to HLB) were inoculated with Flagellin 22 (flg22), a 22 amino acid sequence conserved in the N-terminal part of the bacterial flagellin protein and a PAMP. We used a synthetic peptide based on the available sequence for the flg22 from CLa to assay these plants. Tissue samples were collected before inoculation (time 0) and at 6, 24, 72 and 120 hours post infiltration with flg22. Total RNA has been extracted from all the samples (3 replicates of each) and we have started the gene expression analysis. The expression levels of citrus defense-associated genes is being performed using comparative Ct real time PCR. Genes associated with SAR and PAMP-triggered immunity (PTI) as well as genes in the salicylic acid and jasmonic acid biosynthetic pathways are being studied.
We have successfully made transgenic Arabidopsis plants for most of the constructs that we made so far. Some of the transformations were made in the corresponding mutant background while others were made in wild type (Col-0) background (due to the lack of corresponding mutants). The presence of the transgenes was confirmed by PCR with gene specific primers in the isolated transgenic plants. We have been in the process of testing disease resistance of these plants to Pseudomonas syringae infection. Besides ctNDR1, our recent test of Arabidopsis overexpressing ctEDS5 also showed a complementation of the eds5-3 mutant with the citrus gene. Transgenic plants with potential enhanced disease resistance will be further selected to obtain homozygotes for additional tests of disease resistance. Such constructs will be preferentially used to transform citrus for citrus disease resistance tests. We continue to characterize citrus transgenic plants transformed with ctNDR1. We confirmed with PCR that 29 independently transformed plants carry the transgene. In addition, we conducted second round of infection with Xanthomonas citri (Xac) and results showed again that citrus transgenic plants overexpressing ctNDR1 were more resistant than untransformed controls. We are growing the plants and prepare them for a HLB test in the future. In the meantime, we have made additional transgenic plants with other citrus SA genes. The transformation is generally conducted with two to four genotypes for each construct because there are significant variations in transformation efficiency and resistance to HLB and citrus canker diseases in different genotypes. Besides transgenic plants overexpressing ctNDR1, we have so far made transgenic plants expressing ctEDS5, ctPAD4, ctNPR1, and ctEDS1, which are in US-802, US-812, US-942, and/or Hamlin background. The presence of these transgenes was confirmed with PCR in some genotypes. More transgenic plants are to be obtained from these transformation events from different genotypes. Additional constructs will be placed in the pipeline of transformation once they are ready. The transgenic plants will be prepared for resistance tests for citrus canker and HLB diseases as having been planned for the ctNDR1 transgenic plants.
The objectives of this project are: 1) to generate transcriptome profiles of both susceptible and resistant citrus responding to HLB infection using RNA-Seq technology; 2) to identify key resistant genes from differentially expressed genes and gene clusters between the HLB-susceptible and HLB-resistant plants via intensive bioinformatics and other experimental verifications such as RT-PCR; and 3) to create transgenic citrus cultivars with new constructs containing the resistant gene(s). First group of samples for RNA-Seq were selected at Picos Farm at Fort Pierce, including three Jackson grapefruit plants (resistant/tolerant) and three Marsh grapefruit plants (susceptible). Total RNA has been extracted from the new flush leaf samples of each of these six citrus plants. The qualified RNAs are being used to construct the library for Illumina sequencing. The second group of citrus samples for RNA-Seq has been generated in greenhouse, including progenies from one resistant pomelo parent plant. These two progenies show distinct phenotypes, but both showed high degree of resistance/tolerance after inoculation with Las-infected psyllids. Samples for RNA-Seq include Las-infected, and chemically-cured Las free propagations from these two plants.
This is a continuing project to find economical approaches to citrus production in the presence of Huanglongbing (HLB). We are developing trees to be resistant or tolerant to the disease or to effectively repel the psyllid. First, we are attempting to identify genes that when expressed in citrus will control the greening bacterium or the psyllid. Secondly, we will express those genes in citrus. We are using two approaches. For the long term, these genes are being expressed in transgenic trees. However, because transgenic trees likely will not be available soon enough, we have developed the CTV vector as an interim approach to allow the industry to survive until resistant or tolerant trees are available. A major goal is to develop approaches that will allow young trees in the presence of HLB inoculum to grow to profitability. We also are using the CTV vector to express anti-HLB genes to treat trees in the field already infected with HLB. At this time, we have about 60 different antimicrobial peptides or RNAi constructs are under test against HLB. Plant infected with the CTV vector plus a peptide or RNAi sequence are being inoculated by HLB in a psyllid containment room.
October update. The objectives of this project are 1- Detect the receptors in the insect vector and ligands in the bacterial pathogen, 2- Identify the receptors and ligand, 3- Express receptors/ligands in citrus to block the transmission by psyllids (using CTV-based vector/transgenic plants). In the last few months, we have established the Far-western method (protein overlay assay) in order to detect and consequently identify the receptors in the insect. the big challenge was the unavailability of CLas in culture. We were successful in using infected citrus phloem sap to overlay the insect proteins after separating in PAGE and transfer to PVDF membranes. we used several antibodies produced against Clas. interestingly, we could detect about seven receptors in the entire insect (total proteins). Right now we try to use the dissected guts from insect to know how many of the seven receptors are located in the gut epithelial cells. On the other hand, we established the method to be used with 2D gel electrophoresis. using the 2D gel electrophoresis to separate the insect proteins is very important to validate the number of receptors and to identify the proteins by cutting the spots and analyzing them by MALD-TOF. we expect to have the receptor identified by the next report.
In this work we are examining how the efficiency of HLB transmission by psyllids varies depending on the stage of infection and plant development. One of the questions is what types of flushes are more susceptible to psyllid inoculation with the HLB bacteria. We are using sweet orange and grapefruit plants that have young growing flushes and plants that have only matured flushes. These plants have been exposed to HLB-infected psyllids. Leaves on which psyllids fed were analyzed by PCR to see if the HLB bacterium could be detected soon after the exposure of leaves to infected psyllids. As a result in these experiments, we were able to detect presence of the bacterium fairly early after the initial exposure, approximately after one month. Plants exposed to infected psyllids have been transferred to greenhouse and further monitored for the development of infection. We have repeated this experiment several times and now are analyzing and comparing infection rates of plants with young flushes versus plants with only matured flushes. According to our preliminary data, both young and mature flushes could be inoculated by psyllids, yet inoculation efficiency of mature flushes is lower. In order to characterize potential inoculum sources of the bacterium available for psyllids within an infected tree, we are evaluating the proportion of psyllids that acquired the bacterium after their exposure to different types of flushes during infection development and their ability to transmit infection to new trees. We conducted several trials in which healthy psyllids were placed on either a young growing flush or an older symptomatic flush of an infected tree. Psyllids were secured on those flushes by using small traps made up of mesh material and after 21 days psyllids were analyzed by PCR with HLB-specific primers. Data from PCR analyses demonstrated that Las-positive psyllids were collected from both types of flushes. Psyllids that acquired bacteria from different flushes were next transferred onto healthy receptor plants. These plants are being monitored for the development of infection. Analysis of numbers of plants that became infected upon inoculation with psyllids fed on different types of flushes revealed that more receptor plants that were inoculated by psyllids kept on young flushes became infected and less proportion of receptor plants inoculated with psyllids kept on old flushes became infected with HLB. More trials are in progress. The next objective is to examine psyllid transmission rates from and to citrus varieties that are highly tolerant to HLB. We have propagated 6 different varieties of citrus: Valencia sweet orange, Duncan grapefruit, Persian lime, Eureka lemon, Carrizo citrange, and Poncirus trifoliata. Those varieties represent plants with different degrees of susceptibility to HLB. Currently these plants are being exposed to HLB-infected psyllids. After 1-month exposure, plants were moved to greenhouse and monitored for the development of HLB infection. We are analyzing infection rates for these varieties and intend to use the infected plants as inoculum donors to examine psyllid transmission to new plants.
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