‘Sun Chu Sha’ and ‘Nagami’ kumquat plants were pre-treated with Candidatus Liberibacter asiaticus flagellin 22 (CLas-flg22) peptide 24 hours prior to inoculation with Xanthomonas citri pv citri (Xcc) in an attempt to determine whether CLas-flg22 was capable of inducing PAMP-triggered immunity (PTI). The Xcc population/concentration in the inoculated leaves was determine via colony forming units (cfu) by a standard procedure in a time course of up to 2 days post inoculation (dpi). Bacterial growth in the CLas-flg22 pretreated leaves was lower than in those treated with water in both Nagami and Sun Chu Sha, indicating that PTI was triggered. We used Xcc instead of CLas because of the impossibility of infiltrating the latter pathogen. Similar experiments using Xcc-flg22 also triggered PTI but to much higher levels than CLas-flg22. In other words, bacterial growth was slowed much more.
This is a new project that only just was funded, so there is not yet much to report. However, we do have some preliminary results. Cell penetrating peptides (CPPs) are small protein fragments that have been shown to be able to pass through the cell membrane that surrounds mammalian cells. More significantly, when the CPPs translocate in this manner they can also escort ‘cargoes’ across the membrane. Cargoes include proteins, plasmid or linear DNA, RNA, and antibodies that cannot enter the cell or blood-brain barrier without the presence of CPPs. CPPs have also been shown by others to work to introduce cargoes into plant cells. We have determined what CPPs work effectively in citrus for the import of proteins and nucleic acids. Imported DNA clones transiently express marker proteins; experiments on stable transformation have begun.
The transformation construct for expressing the FLT-antiNodT fusion protein in citrus is nearing completion. We encountered major difficulties cloning the FLT-antiNodT expression cassette into the pTLAB21 citrus transformation vector. The FLT-antiNodT cassette DNA appeared to be unstable in E. coli when cloned into pTLAB21, which stymied our cloning efforts for several months. The instability of the FLT-antiNodT cassette in pTLAB21 was surprising, since the FLT-antiNodT cassette was stable in E. coli when cloned into non-transformation vectors such as pBluescript. For reasons unknown, the FLT-antiNodT cassette was specifically unstable in pTLAB21. However, we serendipitously discovered that the inclusion of an additional segment of DNA next to the FLT-antiNodT cassette in pTLAB21 actually stabilized the FLT-antiNodT cassette in pTLAB21. This piece of DNA was derived from the original FLT-antiNodT cassette cloning vector, pBluescript. This additional piece of DNA should not cause a problem for transformation of citrus or expression of the FLT-antiNodT antibody in transgenic citrus. We are now in the process of sequencing and verifying the pTLAB21-FLT-antiNodT transformation vector. Once this process is complete, we will commence citrus transformations. We anticipate that transformations will begin within the next reporting period.
In the period between April and July of 2013, Citrus Core Transformation Facility (CCTF) experienced significant changes in personnel. These changes reflected negatively on the productivity and as result, the goal of 100 transgenic plants per quarter was not reached. The experimental efforts were directed towards orders that were placed within previous six months. Produced transgenic plants were all Duncan grapefruit. They belong to following orders: MED16-11 plants; X20-four plants; X16-three plants; X19-one plant; X4-one plant; Bi121-six plants; N5-31 plants; N7-nine plants; HGJ3-one plant; and W14-one plant. As opposed to a few previous quarters when the number of placed orders was unusually high, facility received three orders during this time. The ‘genes’ of interest in those vectors were: gMOD1, ELP3-CIV, and ELP4-CIV. All three orders require transformation of Duncan grapefruit plants. Actually, two more orders were placed but after initial attempts to mobilize binary vectors into appropriate Agrobacterium strains the quality of cultures was reviewed and client decided to withdraw them. Within the first year (14 months) of funding of this project, CCTF received 35 orders for production of transgenic plants. Altogether, about 430 plants were produced. They belong to 23 orders received within this funding period and eight orders from previous period. Transgenic plants that were requested from clients in new orders were mostly Duncan grapefruit, but there were some requests for Valencia oranges, Carrizo citranges, and Mexican limes. Ratio of produced plants reflects well the ratio of requested plants in placed orders. CCTF remained a reliable producer of transgenic material. High influx of orders speaks of the important role that CCTF plays in efforts directed towards disease tolerance improvement of Citrus cultivars. Researchers that have well developed ideas or initial encouraging data about the beneficial activity of certain genes know that CCTF can help them test their hypotheses by producing citrus plants that carry those genes in them. While waiting for transgenic material from CCTF, those research groups can direct their efforts towards development of appropriate challenge tests that will be performed on transgenic plants or work on other aspects of protection against citrus diseases.
The project was extended with no cost for an additional nine months in order to conduct the final destructive sampling during the dismantling of the hydroponics greenhouse experiment, and to collect the final year of 2012/13 fruit yields from the ‘Hamlin’ and ‘Valencia’ field trials at Orange Hammock.
Field experiments with nutritional and other horticultural management impacts on HLB disease: Twenty “Hamlin” trees being treated with a comprehensive and holistic HLB management program, including foliar micro and macro nutrients and psyllid control, were harvested in December 2012 for the fifth consecutive year. Half of the trees (10) were symptomatic for HLB (PCR+) prior to the first harvest five years before and the other half were non-symptomatic (PCR undetermined; healthy). The five consecutive harvests show conclusively that HLB, when managed properly, does not kill trees in 2 to 3 years as commonly reported in the literature. Also, the yield of the trees has remained statistically unchanged for the five years of the project, demonstrating conclusively that HLB can be managed and the profitable yields of trees infected with CLas can be sustained. For the 2012-13 harvest season, HLB-affected trees yielded an average 179 lbs of fruit per tree and healthy trees yielded an average 533 lbs of fruit per tree. Furthermore, the juice from all of the fruit (healthy, symptomatic and asymptomatic) has been and continues to be of superior quality, meeting or exceeding USDA standards for Grade A pasteurized not- from-concentrate juice. We state again, although HLB-affected trees have lower per tree yields than healthy trees, their yields are not declining and are statistically similar today as they were five years ago. It is important to note that the added nutrition has not produced a rampant, uncontrolled spread of HLB as evidenced by the healthy trees still producing very high yields. This is because psyllid control in the grove is excellent and the psyllid is the vector of the disease, not nutrition. The grove also shows no evidence of other serious pests and diseases impacting the root systems of trees, such as sting nematodes, Phytophthora and Diaprepes root weevils. HLB is most successfully managed with intensive horticultural practices in the absence of other pests and diseases, many of which don’t have a viable long-term solution. Multiple pest and disease problems superimposed onto HLB-affected trees are likely to result in their rapid decline and death.
Field experiments with nutritional and other horticultural management impacts on HLB disease: Twenty “Valencia” trees being treated with a comprehensive and holistic HLB management program, including foliar micro and macro nutrients and psyllid control, were harvested in April 2013 for the fifth consecutive year. Half of the trees (10) were symptomatic for HLB (PCR+) prior to the first harvest five years ago and the other half were non-symptomatic (PCR undetermined; healthy). The five consecutive harvests show conclusively that HLB, when managed properly, does not kill trees in 2 to 3 years as commonly reported in the literature. Also, the yield of the trees has remained statistically unchanged for the five years of the project, demonstrating conclusively that HLB can be managed and the profitable yields of trees infected with CLas can be sustained. For the 2012-13 harvest season, HLB-affected trees yielded 1.9 boxes of fruit per tree and healthy trees yielded 3.9 boxes of fruit per tree. The juice from the fruit is awaiting analysis; however, for the previous three years the juice from all fruit (symptomatic, asymptomatic, healthy) has met or exceeded USDA standards for Grade A not-from-concentrate juice and based on samples tasted during juicing we do not anticipate a different result this season. We state again, although HLB-affected trees have lower per tree yields than healthy trees, their yields are not declining and are statistically similar today as they were five years ago. It’s also important to note that the added nutrition has not lead to a rampant, uncontrolled spread of HLB as evidenced by the healthy trees still producing above-average yields. This is because psyllid control in the grove is excellent and the psyllid is the vector of the disease, not nutrition.
This project was initiated May 1 2013 and we have focused our efforts on Activity 1. In this activity we are using a rational design strategy to develop and rapidly test individually the two components of a chimeric antimicrobial protein (CAP) based therapeutic that could provide broad spectrum resistance to young citrus plantings against a range of biotrophic bacterial pathogens (HLB and CBCD). Gene mining bioinformatics tools are being used to search existing citrus genomic DNA sequence information available in Phytosome (http://www.phytozome.net) to identify structurally relevant candidate citrus components that can be fashioned into a CAP molecule. The current and highly functional CAP design described by us (Dandekar et al., 2012 PNAS 109(10): 3721-3725) is being used as a scaffold/guide to search for the citrus components. Working with the developer of a recently described bioinformatic tool CLASP we have successfully searched using the active site architecture (3D shape) of the forst component HNE and have found a citrus PR14a sequence whose shape is similar. We have determined the similarity of the citrus protein shape to that of the tomato PR14 and grapevine PR14a proteins. We used the tomato sequence as the protein 3D structure has been determined and could thus be used to thread the citrus and grapevine protein sequences whose structure is yet to be determined. We have also been working on the finding the citrus DNA sequence to replace CecropinB component. We have identified several candidates and are evalusting the similarity in their structure with respect to the known structure of Cecropin B. We have begun the process of designing the vector system to express the citrus PR14a sequence that we have identified. This vector system will be used for the plant based expression system described in our Activity 2. Once designed and constructed will be expressed in Nicotiana benthamiana to produce active protein that can be tested for activity. We have also submitted an application to APHIS-PPQ for the movement of the recently cultured Leberibacter crescens (Lcre). Once we get permission then later this year we can test these proteins for their efficacy using the Lcre to evaluate growth and mortality.
109,913 I did not receive the fund (and hence could not start the project) by this time because of the prolonged negotiation between UCR and CRDF on the intellectual property terms in the contract of this project.
Obj 1A: To increase database mining capabilities the single (sTCW) [C. Soderlund, W. Nelson, M. Willer and D. Gang. (2013) TCW: Transcriptome Computational Workbench; PLOS ONE: accepted for publication] and Multi-TCW (mTCW) psyllid transcriptome databases have been updated and finalized prior to the public release of the database. Now, the Gene Ontology categories (sTCW) can be organized by the number of differentially expressed transcripts (GOSeq, EdgeR) they contain using pairwise comparisons of the different libraries. The addition of statistical software (EdgeR, Pearson’s Correlation Coefficient) to the mTCW_ht_WN2 database allows for key word searches and differential expressional analyses amongst the clusters which will allow for better identification of candidate effectors. To date, the expression of transcript members from 3 clusters shown to be significantly differentially expressed in response to Liberibacter have been validated in both ACP and POP by RT-PCR using primers designed from conserved regions. Obj 1B: Yeast-2 hybrid studies were initiated to study protein-protein interactions important in psyllid-Liberibacter interactions to find key players involved in the circulative, propagative pathway. Previously it was reported that 6 CLas candidate genes had been identified and were in various stages (PCR, cloning, sequencing, etc.) moving towards Yeast 2 Hybrid (Y2H) mating experiments using the ACP gut library and the ACP salivary gland library. That list now contains 19 CLas candidate genes all ready for mating experiments except for; 2 dropped out as unclonable (without a lot of extraordinary measures), 2 cloned and waiting on sequence verification. To date 9 gut library matings and 6 salivary gland library matings have been performed. Data analysis has been completed for 5 of those experiments with the remaining being in various stages moving towards completion (each mating experiment from mating to complete data analysis takes about 18-21 days minimum so stages of completeness are staggered). Through those 5 experiments we have thus far discovered several ACP gene products (‘prey’) that have various levels of interest for making them good candidates for RNAi knock down. The first good RNAi candidate emerged from the mating experiments using both the gut library and the salivary gland prey libraries. The ‘bait’ in this case was CLas-OmpA (Outer membrane protein A). Both ‘prey’ ACP gene fragments that interacted with the ‘bait’ show similarity to integral membrane proteins and could possibly play a role in adhesion or invasion. The next two good candidates came out of the mating experiment again with CLas-OmpA as ‘bait’ and the salivary gland library. The first ‘prey’ insert showed similarity to extracellular matrix-related protein possibly indicating some role with either adhesion or biofilm formation. The second ‘prey’ candidate has domain similarity to a novel component involved in activating signaling cascades in the innate immune response. The fourth and final good candidate to date comes out of a mating experiment between the gut library and CLas-MviN as ‘bait’. MviN has been identified in a variety of bacteria including pathogens and non-pathogens and plant-symbionts. It has been shown to be a virulence factor in Salmonella and is require for motility in Rhizobium. The ‘prey’ candidate pulled out has domain similarity both a protease inhibitor protein (invasion defense) and a putative collagen-binding domain of a collagenase. These four candidates will be moved into the RNAi phase of the project if they fit all criteria specific to RNAi. Obj 2: To date, good quality dsRNA has been made for four psyllid genes predicted to be involved in cytoskeleton formation, defense response, vesicle transport or transcytosis. Feeding studies have been conducted for the gene putatively involved in cytoskeleton formation. Quantitative PCR analysis of psyllids fed on either dsRNA of target gene or buffer showed that there was roughly 20-30% down-regulation of target gene. Results from the transmission experiments performed with this first gene showed subtle differences in the transmission efficiency between plants fed on by dsRNA-treated and control psyllids at 82% and 100% respectively.
The heat-treated trees are continually being monitored for physiological and visual changes. Physiological tests include stomatal conductance readings, water potential readings, and leaf anatomy samples. Pictures are taken monthly to monitor the tree’s recovery from the heat treatment as well as to monitor any signs of reinfection. Recently, more tests have been done to assess the effectiveness of the heat treatment. Average fruit diameter, average fruit set, average leaf size, and average leaf area index (LAI) were measured in the 36 treated trees as well as 9 control trees for comparison. Physiological indices: average fruit diameter, average fruit set, and average leaf size can show how the heat treatment has affected the disease as well as the tree itself. If the bacteria are successfully reduced or eliminated, the physiological indices should increase after treatment. The average LAI quantifies the leaf density of the tree canopy. In a HLB-infected tree, LAI decreases as the disease progressed. The data from these tests are currently being analyzed. The chlorophyll fluorescence is also being measured. This measurement is a good indicator of overall tree health. The cross-sections of leaf petioles have been returned from the lab and are currently being stained for analysis. Once staining is completed, the phloem area can be measured. The phloem cells of an infected citrus tree tend to collapse, causing the overall phloem area to be reduced and blocking nutrient transport throughout the tree. Examining the phloem area before and after treatment could provide more evidence to support the success of heat treatment.
Data have been collected from five growers. We are developing and evaluating models for Valencia and Hamlin oranges at low and high levels of HLB.
For this quarter, we continue data collection on the trial set-up in February 2010 to evaluate the interaction between nitrogen and hedging date. The field trial was a factorial experiment with 3 nitrogen levels (0, 50 and 100 lb N/ac) and three hedging treatments namely non-hedged trees, early hedging in February, and late hedging in April. These 9 treatments were split into subplots with one subplot sprayed with a recommended insecticide at the beginning of the flush cycle for psyllid control and the other subplot left unsprayed. One spray application was done during this quarter with imidacloprid (Provado at 20 oz/ac) just prior to the major flush cycle of early July. We continued the monitoring of various developmental stages of psyllids on flush shoots. During this quarter, all non-hedged trees at different nitrogen levels and with no pesticide application harbored significantly more psyllids than the other treatment. Flush production during the July flush cycle was more important in non-hedged trees relative to trees hedged in February and April, in contrast to what was observed in the previous flush cycle in May. Relative to non-fertilized trees, trees that received nitrogen had significantly more psyllids. Specifically, the non-hedged trees that received nitrogen had 3 to 5-fold more psyllids than the non-fertilized ones. Application of imidacloprid to trees was very effective at controlling psyllid populations independently of hedging or nitrogen treatment. Hardly any psyllid eggs, nymphs or adults could be collected from all plots that received Provado treatment.
Due to its reduced genome, Candidatus Liberibacter asiaticus has only a few transcriptional regulators. We hypothesized that natural and/or synthetic chemicals can interact with these transcription factors to interfere with regulatory activities, resulting in decreased tolerance to the stress conditions encountered in the phloem. During the first year of this proposal, we focused on the identification of small molecules that bind and/or modify Ca. L. asiaticus transcriptional regulators. Each regulator was cloned, purified, screened against a chemical library of 1200 small molecule. We identified several small molecules that interact with CLIBASIA_01180 and CLIBASIA_01510. Thermal denaturation experiments were then performed with each of the identified small molecules, at numerous concentrations, to confirm the effects of these ligands in vitro.. The second year of this proposal focused on the in vitro and most importantly, in vivo experiments, to prove the specificity of the chemicals identified. For CLIBASIA_01510, seventeen chemicals were identified that increased the mid-transition temperature during thermal denaturation assays. CLIBASIA_01510 is homologous to proteins involved in gene regulation through direct interaction with the beta subunit of the RNA polymerase, not by direct binding to DNA. Based on these observations, the effect of chemicals identified in the screen, were tested in vivo using a bacterial two-hybrid system. We also determined the optimal growth media and growth phase to obtain tight protein:protein interactions. We then examined the effects of each ligand. Two chemicals that were identified during the chemical screens were found to disrupt the CLIBASIA_01510:RpoB interaction. Further experiments, using the chemical scaffold as a guide, identified another chemical that binds to the complex with higher affinity. Using Liberibacter crescens, we found that the administration of the chemical decreased mRNA expression of CLIBASIA_01510 homologs. The decrease in mRNA expression was positively correlated with decreased stress tolerance. The protein CLIBASIA_01810 is a transcriptional regulator that modulates proteins involved in cell wall synthesis. During year 2 of this project, our primary focus was testing the effects of the identified chemicals on the survival of Ca. L. asiaticus in infected orange seedlings. These experiments were performed using leaves from 18 month old infected seedlings (kindly provided by Dr. Svetlana Folimonova at Lake Alfred). To this end, a new protocol for the efficient extraction of Ca. L. asiaticus mRNA was established. Brief exposure to the selected chemicals substantially reduced the metabolic capacity of Ca. L. asiaticus. We observed a significant decrease in the transcription of genes regulated by CLIBASA_01810, as well as a decrease in the abundance of 16S RNA genes. The effects of each chemical were confirmed in L. crescens, where modifications to the cell wall were found to lower tolerance to stress conditions. These exciting results suggest longer periods of treatment could result in the complete elimination of the pathogen.
The Core Citrus Transformation Facility (CCTF) continues to serve the community of researchers exploring ways to improve Citrus plants and make them tolerant/resistant to diseases. CCTF does its service by producing transgenic material. Within the last quarter, the CCTF facility worked on producing transgenic plants of the following combinations: produced the following transgenic citrus plants (transgene in parenthesis): Mexican lime (pHK vector); Duncan grapefruit (ELP3 gene); Duncan (MKK7 gene); Duncan plants (p7 gene); Duncan (p10 gene); Mexican lime and Hamlin (p33 gene); Duncan (SUC-CitNPR1 gene); Duncan (pWG19-5 vector); Duncan plants (pWG20-7 vector); Duncan (pWG21-1 vector); Duncan (pWG22-1 vector); Duncan (pWG24-13 vector); and Duncan (pWG25-13 vector). All of these plants are for researchers funded by CRDF in the battle against HLB and canker.
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