Obj 1: As a first step to validate bioinformatically predicted effectors identified from mining of our PAVE database, we selected 20 interesting candidates that were detected in both the ACP and PoP transcriptomes. Primers were designed around the most conserved nucleotides in each sequence and RT-PCR was performed using PoP cDNA. To date, 12 of those transcripts are confirmed to be expressed. Confirmation of the remaining 8, as well as all genes in ACP by RT-PCR is ongoing. Obj 2: To initiate yeast-2 hybrid studies to elucidate protein-protein interactions important in HLB disease, 500 guts from Las-infected ACP, and 500 guts and 1000 salivary glands from uninfected ACP have been isolated. Good quality RNA has been isolated from all of these organs and cDNA library preparation is underway. Obj 3: For proteomic identification of putative effectors proteins from 50 guts and 250 salivary glands of infected and noninfected potato psyllid adults were separated by SDS-PAGE (10’14% acrylamide). After trypsin digestion, peptides were analyzed by nanoLC-LTQ-Orbitrap (Thermoelectron) mass spectrometry. We identified 828 and 358 proteins in the gut using all the PoP and ACP translated PAVE databases, respectively. Using a 2 fold change cut-off, 45% of the proteins identified using the PoP transcripts (373/828) and 40% of proteins using the ACP transcripts (142/358) showed differential abundance in the uninfected proteome compared to the Lso-infected proteome. We identified 729 and 357 proteins in the salivary gland using all the PoP and ACP translated PAVE databases, respectively. Using a 2 fold change cut-off, 58% of the proteins identified using the PoP transcripts (425/729) and 55% of proteins using the ACP transcripts (198/357) showed differential abundance in the uninfected proteome compared to the Lso-infected proteome. Obj 4: Using data from objectives 1 and 3 (above), we have selected two psyllid and two Liberibacter genes for functional characterization by RNAi analysis using the oral delivery method. Good quality dsRNA has been made using the MEGAscript RNAi kit and feeding studies are underway. In addition to qPCR validation of mRNA knockdown using the psyllid COI gene for normalization, we have designed novel bioassays to confirm predicted functional roles in the transmission process. Psyllids can survive up to 2 weeks in buffer only controls. Selection of salivary gland-specific genes for RNAi analysis by microinjection is underway. We have optimized the injection procedure with a novel pre-puncturing system using a fine tungsten wire followed by insertion of fluid-filled needle. Currently, psyllid survivability on buffer only controls is 50% and we hope to improve upon this with by exploring modifications.
In this project, we proposed three aims in order to identify, characterize, and make use of citrus genes with a potential role in SA-mediated defense in engineering resistance to canker and greening diseases in citrus plants. Among the three proposed aims, the first aim has been completed, the second aim is about to be finished, and the third aim needs longer time to complete due to long-term growth nature of citrus plants. So far we have identified at least one citrus SA gene that could have effects on canker disease when overexpressed. Additional citrus transgenic plants are under further production and defense tests. We believe that we have met the expectations of the project and here provide a summary of the project. Objective 1: Identify genes positively regulating SA-mediated defense in citrus We identified over 10 citrus SA homologues via bioinformatics analysis. We used an RT-PCR approach to clone 10 full-length cDNA for the citrus SA homologues, which were further cloned into a binary vector pBIN19ARSplus for making transgenic plants in Arabidopsis and citrus. We have also finished collecting citrus tissues infected with Ca. L. asiaticus in a time course. qRT-PCR analysis with these samples was conducted for some SA genes. Our results showed that expression of at least one of the genes, ctNDR1, showed an induction upon HLB infection, suggesting a possible role of ctNDR1 in defense against HLB. Objectives 2: Complement Arabidopsis SA mutants with corresponding citrus homologues All 10 SA citrus genes were used to transform Arabidopsis plants, either complementing the corresponding mutants or overexpressing in wild type. We obtained T0 seeds for these constructs and selected most T0 seeds for T1 transgenic plants. Some seeds were further selected for homozygotes at the T2 generation. Most of the transgenic plants were tested for disease resistance to the infection of Pseudomonas syringae. So far, we found that at least two of the constructs ctNDR1 and ctEDS5 showed some level of disease resistance. However, there was no significantly increased resistance in CtNPR1 transformed Col or npr1-1 mutant and CtPAD4 transformed Col or pad4-1 mutant. Additional tests are undergoing for other transgenic plants. We have done more detailed characterization of ctNDR1 plants, which was summarized in a previous progress report (April 2012). A manuscript for this work should soon be submitted for a consideration of publication. Objectives 3: Assess the roles of SA regulators in controlling disease resistance in citrus We have so far produced transgenic plants for ctNPR1, ctEDS5, ctPAD4, and ctNDR1 and the presence of the transgenes in these plants were confirmed by PCR. In addition, we have tested disease resistance of ctNDR1 plants with Xanthomonas citri subsp (Xac), the causal agent for citrus canker disease, and found that overexpressing this gene confers some level of resistance to the strain. We will further test if ctNDR confers resistance to greening disease. In addition, we will continue to produce transgenic plants overexpressing other SA genes and selected transgenic plants will be tested for resistance to canker and greening diseases. These activities will be conducted after the end of the grant period.
In this project, we proposed three aims in order to identify, characterize, and make use of citrus genes with a potential role in SA-mediated defense in engineering resistance to canker and greening diseases in citrus plants. Among the three proposed aims, the first aim has been completed, the second aim is about to be finished, and the third aim needs longer time to complete due to long-term growth nature of citrus plants. So far we have identified at least one citrus SA gene that could have effects on canker disease when overexpressed. Additional citrus transgenic plants are under further production and defense tests. We believe that we have met the expectations of the project and here provide a summary of the project. Objective 1: Identify genes positively regulating SA-mediated defense in citrus We identified over 10 citrus SA homologues via bioinformatics analysis. We used an RT-PCR approach to clone 10 full-length cDNA for the citrus SA homologues, which were further cloned into a binary vector pBIN19ARSplus for making transgenic plants in Arabidopsis and citrus. We have also finished collecting citrus tissues infected with Ca. L. asiaticus in a time course. qRT-PCR analysis with these samples was conducted for some SA genes. Our results showed that expression of at least one of the genes, ctNDR1, showed an induction upon HLB infection, suggesting a possible role of ctNDR1 in defense against HLB. Objectives 2: Complement Arabidopsis SA mutants with corresponding citrus homologues All 10 SA citrus genes were used to transform Arabidopsis plants, either complementing the corresponding mutants or overexpressing in wild type. We obtained T0 seeds for these constructs and selected most T0 seeds for T1 transgenic plants. Some seeds were further selected for homozygotes at the T2 generation. Most of the transgenic plants were tested for disease resistance to the infection of Pseudomonas syringae. So far, we found that at least two of the constructs ctNDR1 and ctEDS5 showed some level of disease resistance. However, there was no significantly increased resistance in CtNPR1 transformed Col or npr1-1 mutant and CtPAD4 transformed Col or pad4-1 mutant. Additional tests are undergoing for other transgenic plants. We have done more detailed characterization of ctNDR1 plants, which was summarized in a previous progress report (April 2012). A manuscript for this work should soon be submitted for a consideration of publication. Objectives 3: Assess the roles of SA regulators in controlling disease resistance in citrus We have so far produced transgenic plants for ctNPR1, ctEDS5, ctPAD4, and ctNDR1 and the presence of the transgenes in these plants were confirmed by PCR. In addition, we have tested disease resistance of ctNDR1 plants with Xanthomonas citri subsp (Xac), the causal agent for citrus canker disease, and found that overexpressing this gene confers some level of resistance to the strain. We will further test if ctNDR confers resistance to greening disease. In addition, we will continue to produce transgenic plants overexpressing other SA genes and selected transgenic plants will be tested for resistance to canker and greening diseases. These activities will be conducted after the end of the grant period.
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. We have obtained the sequences from five of the six samples; a total of ca. 269 millions reads and ca. 49 GB nucleotide sequences were just obtained, and we are going to conduct intensive bioinformatic analysis from now on. While waiting for the sequences, we conducted a genome wide prediction of citrus resistance genes using genome annotated genes and unigenes of Citrus clementine (Cc) and C. sinensis (Cs) from Citrus Genome Database. We identified 607, 484 and 499 genes containing the NBS domain (NB-ARC, PF00931) in C. clementine, C. sinensis and Csc respectively using pfam_scan. There were 426, 250 and 322 genes after filtering with NBS domain coverage over 80% (230 bp over 287 bp of NB-ARC domain). There are more NBS-containing proteins in the genome of C. clementine than that of C. sinensis. Some of the NBS genes were found to have an expression. For C. clementine and C. sinensis, there were 118,381 and 214,858 mRNAs or ESTs deposited in GenBank and 93 out of 607 and 221 out of 484 NBS related genes match one or more ESTs respectively. The number of EST varied from 1 to 25. We predicted the NBS orthologs of citrus with inparanoid and multiparanoid. A total of 131 orthologs were identified, which contains 213, 146 and 191 NBS genes for C. clementine, C. sinensis and Csc, respectively. A total of 241 NBS genes were specific to C. clementine. Although the total number of NBS genes were similar in the two varieties of sweet orange, there were 226 and 198 variety-specific NBS genes assigned to each, respectively. A total of 318 out of 499 NBS genes in sweet orange could be mapped to the 9 chromosomes. The NBS genes clustering distributed in chromosome 1, 3 and 5 in sweet orange. There are still 181 NBS genes that could not be mapped to the known chromosomes.
The goal of this project is to develop a transcriptomic toolbox to elucidate psyllid-Ca. Liberibacter (Las and Lso) interactions and associated effectors that can be used for management of citrus greening disease. In this project we constructed 14 Illumina paired-end libraries including uninfected adults, infected adults, uninfected nymphs, infected nymphs, uninfected guts, infected guts, uninfected salivary glands and infected salivary glands generating 72,539 and 81,682 unique transcripts (unitrans) from Asian citrus psyllid (ACP) and potato psyllid (PP), respectively. Secondly, we developed a user-friendly database containing the assemblies ‘DcWN’ and ‘BcWN’ from the eight libraries yielding a total of 45,799 and 81,682 unitrans. Of the 45,799 total unitrans, 17,988 unitrans (39%) were thus annotated, which comprised 35.6 Mb of sequence with an average unitrans length of 1,981 bp, ranging from 150 bp to 26,540 bp. Of the 81,682 total unitrans, 16,462 unitrans (20%) were annotated. Unitrans resources comprise 28.9 Mb of sequence with a N50 length of 1,390 bp and an average unitrans length of 1,756 bp, ranging from 100 bp to 27,405 bp. Comparison of expression data between the infected and uninfected Asian citrus psyllid highlights several differences. We have also been able to identify genes involved in RNAi based silencing. Results from comparative transcriptomic analyses showed that ACP and PP are more similar than dissimilar, and suggesting that researchers in quarantined areas without access to ACP can use the PP as a surrogate model organism to advance research efforts. To our knowledge this is the first report elucidating a detailed transcriptome analysis of ACP and PP. The data provides insights about vector-pathogen relationships at the molecular genomic and gene expression levels. This effort provides an invaluable resource to aid in effector gene identification relevant to establishment and transmission pathways in the psyllid host. Using the databases, putative gene families common to both psyllids and also other insect genomes were identified, as well as a suite of unique sequences that when taken together with the Ca. Liberibacter genome sequences can make possible predictions about ‘interactors’. In addition a pattern of responses to Ca. Liberibacter infection indicated that that Ca. Liberibacter infection negatively affects psyllid nymphs to a higher degree than adults. Additionally, transcriptomic data show it can be used to study obligate endosymbionts based on presence of transcripts sharing high nucleotide sequence similarity to Wolbachia sp. and Carsonella. In silico gene expression analyses identified several psyllid and bacterial genes putatively involved in psyllid development, Ca. Liberibacter transmission and pathogenesis (adhesion, nutrition, pathogenicity, lytic function, viral functions). Based on differential expression patterns of specific gene ontology functions such as iron metabolism and immune responses, we identified the candidates transferrin and Caspar, that could be investigated further to understand their predicted roles in transmission and bacterial survival. Also we identified differentially expressed transposon-like transcripts in infected and uninfected libraries. Several transposable element-related transcripts are differentially expressed in the Ca. Liberibacter infected libraries that may be significant by aiding Ca. Liberibacter a competitive advantage over the psyllid host. Experiments to validate candidate ‘interactors’ and test putative transcript functionality are underway using various strategies including transcript knockouts. We have optimized feeding assays for dsRNA delivery, and qPCR analysis of gene expression of gut genes, and are optimizing microinjection as the next step for monitoring knockouts directed at salivary gland/oral box-secretome effectors.
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.
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