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.
The main objective of this proposal is to find ways to optimally deploy the superinfecting Citrus tristeza virus (CTV)-based vector to prevent existing field trees from development of the HLB disease and to treat trees that already established the disease. This is a new project and the funds for this project were released. We already have designated personal for conducting the proposed research. The research is in progress. Plant material that will be used in this project is being prepared in our greenhouse. Using plant material and inoculum sources that are already available, we are conducting initial experiments to examine the levels of multiplication of the superinfecting CTV vector in trees infected with different field isolates of CTV. Young citrus trees are being inoculated with various isolates of CTV. Upon establishment of the initial infections the trees will be inoculated with the green fluorescent protein (GFP)-marked CTV vector. Levels of vector expression (monitored based on GFP expression) will be evaluated. The objective here is to investigate how previous infection of trees with the virus affects the ability of the vector to infect and multiply in those trees. The second objective is to examine the effect of various rootstock/scion combinations on the superinfecting ability of the vector in order to evaluate what combinations would support high levels of vector expression. Currently we are preparing plants that have different rootstock/scion combinations that will be used for the experiments proposed under this objective. Both first and second objectives represent our main focus for the first year of funding.
A transgenic test site has been prepared at the USDA/ARS USHRL Picos Farm in Ft. Pierce, to support HLB/ACP/Citrus Canker resistance screening for the citrus research community. There are numerous experiments in place at this site where HLB, ACP, and citrus canker are widespread. The first trees have been in place for almost three years. Dr. Jude Grosser of UF has provided 550 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser planted an additional 89 trees including preinoculated trees of sweet orange on a complex tetraploid rootstock that appeared to confer HLB resistance in an earlier test. Dr. Kim Bowman has planted several hundred rootstock genotypes transformed with the antimicrobial peptide D4E1. Texas A&M Anti-ACP transgenics produced by Erik Mirkov and expressing the snow-drop Lectin (to suppress ACP) have been planted along with 150 sweet orange transgenics from USDA expressing the garlic lectin. Eliezer Louzada of Texas A&M has permission to plant his transgenics on this site, which have altered Ca metabolism to target canker, HLB and other diseases. More than 120 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) have been planted in a replicated trial in collaboration with Fred Gmitter of UF and Mikeal Roose of UCRiverside. Plants will be monitored for CLas development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding. Dr. Roose has completed initial genotyping on a sample of the test material using a “genotyping by sequencing” approach. Additional plantings are welcome from the research community.
A number of experiments and observations at the USDA/ARS Ft. Pierce citrus scion improvement program demonstrate resistance or tolerance to HLB in some conventional citrus genotypes. In the funding cycle that began in 2012 our efforts in this area are markedly enhanced by funding for a postdoctoral researcher. Dr. Sharon Inch, who has extensive experience in plant pathology and histology, began working on this project 10/9/2012. She will move forward with established experiments comparing HLB development in specialty cultivars, which displayed resistance in a study of commercial groves, vs. susceptible standards. A set of replicated plants have been initiated for 50 genotypes representing advanced USDA selections, genotypes which display apparent resistance/tolerance in field observations, and susceptible standards: these will be exposed to HLB in a hot psyllid house and will be assessed for CLas levels, HLB symptoms, and growth. The remaining proposed experiments relating to assessing and characterizing HLB resistance/tolerance will be initiated in the next quarter.
The funding cycle that began in 2012 provides for a postdoctoral researcher in the USDA/ARS scion transgenic program. This will markedly enhance progress. Dr. Guixia Hao, who has extensive experience in plant transformation and molecular biology, began working on this project 9/23/2012. She will move forward with new constructs and resulting transgenics, including hairpins to suppress PP-2 through RNAi (to test possible reduction in vascular blockage even when CLas is present), chimeral constructs that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab), a citrus promoter driving citrus defensins (designed by Bill Belknap of USDA/ARS, Albany, CA), and genes which may induce deciduousness in citrus. A series of transgenics scions and rootstocks, produced in the last several years, continue to move forward in the testing pipeline.
In the initial funding of the current grant we have made progress on several of our objectives: Objective 1. Evaluate existing transformed lines: We have been maintaining a steady effort in the growing and testing out of candidate transformed lines from our large transformation efforts. Over 34,000 transformations of Duncan grapefruit have been carried out with 8 different reporter and resistance constructs, from which we have generated over 600 T0 plants that are being used for PCR analysis and pathogen testing. We are currently testing plants. The frequency of functional transformants may be lower than typical given that inappropriate leaky expression will be counter-selected. Objective 2. Expand stable transformations Our efforts to transform additional commercial citrus species has been focused on Ruby Red grapefruit and sweet orange. At present we have introduced eight constructs consisting of promoters with1,4, or 14 TAL effector/PthA binding sites with GUS or resistance gene coding regions in more than 1200 Ruby Red and over 600 sweet orange transformations, producing about 60 T0 plants for analysis. Objective 3. Refine constructs We have initiated additional resistance gene constructs that contain additional promoter elements and/or use another resistance gene known as AvrGf2. As each new construct is completed, we are testing these it in transient and stable transformation assays. Objective 4. Sequence more TAL effectors from additional canker accessions We have new sequences of TAL effectors from strains from Florida, Argentina, and Brazil, including the Miami “type” strain, as well as several strains with altered growth phenotypes. Last, we are expanding efforts at examining effects of resistance gene constructs on population growth of a range of Xanthomonas citri strains.
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