The mature citrus transformation facility continues to produce transgenic events for its clients. Productivity has significantly improved using vectors with reporter genes. Transgenics produced via Agrobacterium can now be supplemented with transgenics produced using biolistics in immature and mature citrus. Scientists have been encouraged to submit vectors with all plant sequences and no pest sequences, which might lessen regulatory hurdles. Genes are now being stacked in an effort to prevent the pathogen from easily overcoming tolerance or resistance. A manuscript is being prepared describing biolistics in immature citrus. Currently, all transgenic events are being transferred to scientists directly without secondary grafting or propagation, unless otherwise requested. To improve efficiencies and lessen potential micrografting incompatibilities, sweet orange is being micrografted onto decapitated sweet orange rootstock. Micrografting losses in mature rootstock have significantly decreased when young shoots are micrografted onto decapitated rootstock grown in high sucrose solution. We are in the process of introducing new breeder lines in which to produce transgenics. Three sweet orange varieties and one grapefruit are being introduced via shoot-tip grafting. Once plants are established, they will be used in budding mature citrus onto rootstock to obtain budstick for transformations. Our website is being updated to reflect current prices and technologies employed.
The overall objective of this project was to develop new girdling techniques capable of stopping or limiting the movement of CLas to the roots while allowing for normal phloem transport, thereby enabling young trees to be more tolerant to HLB in the field. Two girdling patterns were designed differing in the distance photoassimilates had to travel apoplastically (around the girdle) to re-enter the unobstructed phloem. We began experimentation with the girdling design (spiral) that presents the longest distance for apoplastic movement and the longest distance for CLas (or its signal) to travel. To test the hypothesis, spiral girdles were placed on the main stem or on a lateral stem, and trees budded with HLB material above the girdle. In both instances, and within 8 months, approximately 90% of the trees became HLB+ opposite to the girdle. The results are clear indication that CLas (or its signal) is capable of movement laterally across the phloem tube side walls. Based on the convincing results using the girdle pattern, the number of trees to be tested with the checkerboard pattern was reduced. As with the spiral pattern, HLB was transmisible through the severed phloem elements. The data collected in this project offer new insights into the nature of the HLB signal, as it seems to move through physical barriers smaller then the actual size of CLas. We conclude that genetic HLB signal is sub-cellular in nature and can be transferred between non-phloem living cells. This new finding is important in that it shows that HLB causing effector is not necessarily the entire CLas bacterium. The infected HLB trees were kept to be used in current CRDF-sponsored research projects.
The main accomplishments during this quarter: We continued testing the effect of the K gene on transformation efficiency of a lemon cultivar. In general, lemon cutilvars are difficult to be genetically transformed. We have observed the K gene can drastically improve the transformation efficiency of the lemon cultivar used, similar to the effects of the K gene on the other orange cultivars tested. We continued to repeat the effects of K and I genes on transformation efficiency of mature citrus explants of mature Pineapple orange. The effects of the K and I genes have been confirmed. We have also tested effects of a non-conventional regulator of gene expression on regeneration efficiency of Washington navel orange and Valencia orange. We observed several fold increases in shoot regeneration efficiencies of both cultivars. Our goal is to use this regulator and also its combination with the K gene to improve transformation efficiencies of of both juvenile and mature citrus tissues One manuscript reporting the drastically improvement of several citrus cultivars has been submitted in the end of August. We have started to write the second manuscript from the project.
Effectors are essential virulence factors in microbial pathogens. The HLB-associated bacterium Candidatus Liberibacter asiaticus (Las) is known to encode the Sec secretion system, which is predicted to deliver effector proteins into plant phloem. Our previous research identified four Sec-secreted proteins from Las. The goal of this project is to characterize the targets of these effectors in citrus. This research will provide important knowledge on the basic biology of HLB pathogenesis and facilitate the development of control strategies. Our research has been focusing on four Las effectors whose expression can be consistently detected from various citrus varieties that are infected by Las. We hypothesized that their targets in citrus contribute to HLB development. The main approach we are using to identify the effector targets is yeast two hybrid (Y2H) screening. In the past two years of this project, we accomplished the following experiments: 1) expression analysis of the Las effectors; 2) cloning and expression of the effector genes in yeast; 3) construction of a normalized citrus cDNA library with more than 3 millions of primary clones using HLB-infected RNA samples; 4) Illumina sequencing-based Y2H screening using each of the four Las effectors as the bait and sequencing data analysis; 5) subcellular localization analysis of the Las effectors. During this report period, we focused on experimental confirmation of effector targets. From Y2H screening, multiple potential targets were identified for each effector. We performed extensive literature search and prioritized our effort on candidates with potential roles in plant defense or HLB symptom development. These candidates were re-cloned from citrus cDNA individually and examined for their interaction with the corresponding effector by targeted Y2H. Due to the large amount of work, we have further focused on two effectors, which exhibit significantly higher expression (10-40 folds) in HLB-infected citrus tissues than in psyllids. So far, we examined a total of over 20 potential interacting proteins of these two effectors. Our results strongly suggest that each of these effectors specifically interacts with a class of citrus proteins in yeast. Importantly, these two citrus plant families have been reported to play a role in plant defense and they are also well-known virulence targets of other bacterial and fungal pathogens. Furthermore, one of the protein family has been reported to be present in the vascular system. We have made exciting progress in the project. Our on-going effort includes: 1) further confirm the physical interaction of the effectors and their targets using in vitro and in vivo co-immunoprecipitation assays; 2) characterize the target proteins as a family to understand how they interact with the effectors.
During this reporting period (July, August, and September, 2015), the transgenic plants to be developed for this project continued to grow at two different locations in secure greenhouses and growth chambers. Seven independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct are growing in Dr. McNellis’ lab at the Pennsylvania State University at University Park, PA, and an additional eight independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct are growing at Dr. Tim Gottwald’s lab at the United States Horticultural Laboratory in Fort Pierce, Florida. These plants are continuing to be propagated at both Ft. Pierce and Penn State. Our collaboration with Dr. Janice Zale (University of Florida Mature Citrus Transformation Facility, Lake Alfred) to transform varieties important to the Florida citrus industry, including the ‘Valencia’ and ‘Hamlin’ sweet orange varieties and the ‘Citrumello’ and ‘Carrizo’ rootstocks with the FLT-antiNodT expression construct, has had initial success. Hamlin and Carrizo transformants are now growing at Lake Alfred. Dr. Zale will maintain the original transformants, and will send propagated cuttings to Penn State for molecular analysis over the next 3-6 months. We will also send some of the propagated sweet orange and rootstock plants to Ft. Pierce for HLB resistance testing in collaboration with Dr. Tim Gottwald and possibly Ed Stover. During this reporting period, we also initiated development of an FLB-antiNodT expression cassette in the transformation construct pBI121, which has a history of successful approval for transgenic plant development. We anticipate that this construct could be completed during the next reporting period, and we would forward it to Dr. Zale immediately upon completion for further citrus transformations. In August, Dr. McNellis presented a poster at the annual meeting of the American Phytopathological Society in Pasadena, CA, describing the results so far, including successful expression of the FT-scFv protein in grapefruit with minimal or no negative effects on plant phenotype.
During this reporting period (July, August, and September, 2015), the transgenic plants to be developed for this project continued to grow at two different locations in secure greenhouses and growth chambers. Seven independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct are growing in Dr. McNellis’ lab at the Pennsylvania State University at University Park, PA, and an additional eight independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct are growing at Dr. Tim Gottwald’s lab at the United States Horticultural Laboratory in Fort Pierce, Florida. These plants are continuing to be propagated at both Ft. Pierce and Penn State. Our collaboration with Dr. Janice Zale (University of Florida Mature Citrus Transformation Facility, Lake Alfred) to transform varieties important to the Florida citrus industry, including the ‘Valencia’ and ‘Hamlin’ sweet orange varieties and the ‘Citrumello’ and ‘Carrizo’ rootstocks with the FLT-antiNodT expression construct, has had initial success. Hamlin and Carrizo transformants are now growing at Lake Alfred. Dr. Zale will maintain the original transformants, and will send propagated cuttings to Penn State for molecular analysis over the next 3-6 months. We will also send some of the propagated sweet orange and rootstock plants to Ft. Pierce for HLB resistance testing in collaboration with Dr. Tim Gottwald and possibly Ed Stover. During this reporting period, we also initiated development of an FLB-antiNodT expression cassette in the transformation construct pBI121, which has a history of successful approval for transgenic plant development. We anticipate that this construct could be completed during the next reporting period, and we would forward it to Dr. Zale immediately upon completion for further citrus transformations. In August, Dr. McNellis presented a poster at the annual meeting of the American Phytopathological Society in Pasadena, CA, describing the results so far, including successful expression of the FT-scFv protein in grapefruit with minimal or no negative effects on plant phenotype.
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 are continuing to screen possible peptide candidates in our psyllid containment room. We are now screening about 80 different genes or sequences for activity against HLB. We are starting to test the effect of two peptides or sequences in combination. We have developed methods to be able to screen genes faster. Finally, we have found a few peptides that protect plants under the high disease pressure in our containment room with large numbers of infected psyllids. We now are examine combinations of peptides for more activity. We recently examined all of the peptides constructs for stability. The earliest constructs have been in plants for about nine years. Almost all of the constructs still retain the peptide sequences. One of the peptides in the field test remained stable for four years. All of these constructs had the peptide gene inserted between the coat protein genes, which is positioned sixth from the 3′ terminus. However, we have found that much more foreign protein can be made from genes positioned nearer the 3′ terminus. Based on that we built constructs with the peptide gene next to the 3′ terminus. These constructs produced much greater amounts of peptide and provided more tolerance to Las. Unfortunately, they are less stable. So now we are rebuilding constructs with the peptide gene inserted at an intermediate site hoping for a better compromise of amounts of production and stability. We have produced a large amount of inoculum for a large field test via Southern Gardens Citrus. We are screening a large number of transgenic plants in collaboration with Dr. Zhonglin Mou, Department of Microbiology and Cell Science in Gainesville, to test transgenic plants over-expressing plant defense genes. We are propagating a progeny set of plants of the promising candidates for a final greenhouse test.
Evaluation of root production on HLB-affected trees compared to presumed healthy trees confirmed that root loss is due to reduced root longevity. This root loss is exacerbated by biotic and abiotic stresses in the rhizosphere. Phytophthora propagules per soil volume and per root fluctuate in response to fibrous root density based on intensive rhizosphere soil sampling (i.e., local repeated measures) and extensive sampling (i.e., Syngenta statewide Phytophthora propapgule survey) . Increased susceptibility of Las infected roots to Phytophthora spp. was evidenced by statewide populations that fluctuated from unprecedented highs in the 2011 season to an unprecedented low in 2013 compared to 25 years of pre-HLB soil populations. A series of greenhouse studies with potted seedlings investigated the interaction between Las and P. nicotianae (P.n.) The results demonstrated that 1) Las infection of citrus rootstocks predisposes fibrous roots to P.n. infection by increasing root leakage of exudates that attract zoospores and by disrupting host resistance (i.e. carbohydrate-mediated defense); 2) the combination of Las and P.n. causes greater damage than each pathogen alone; 3) the interaction between P.n. and Las on fibrous root damage is mediated by available young fibrous root biomass. More in-depth examination of the interaction between Las and P.n., revealed different disease causation mechanisms for each root pathogen. Las damages fibrous roots by inducing faster growth of replacement roots and root turnover; P.n. damages fibrous roots by causing rapid root collapse immediately after infection, especially new growth; canopy development is reduced after root damage by both pathogens. To explore disease development from the perspective of root carbohydrate metabolism responses to Las and P.n. infection, sucrose metabolism related gene expression was investigated in two rootstocks (Cleopatra mandarin, susceptible to P.n.; Swingle citrumelo tolerant to P.n.). The results showed that sucrose metabolism is more disrupted by Las and P.n. in Phytophthora susceptible Cleopatra mandarin than Swingle citrumelo. Chemical control of P.n. slows infection of the root system and may slow decline in HLB-affected trees in Phytophthora-infested groves. However, greenhouse and fungicide trials indicate that HLB reduces the effectiveness of fungicides for control of Phytophthora root rot as a consequence of increased root susceptibility to P.n. Based on these results, grove managers should consider practices that: 1) minimize root damage caused by the combination of Las and P.n. (e.g. use of soil fungicides if damaging populations of Phytophthora occur); 2) reduce disruption of sucrose metabolism in rootstocks and scions through balanced use of water and fertilizers to promote regular cycles of root and shoot flushes and sustain fruit growth and maturation; 3) provide optimal growing conditions to maintain tree growth by minimizing the effects of abiotic (e.g., drought, freezes) and biotic stress (root pests and pathogens).
This proposal is aimed at following previous work in CRDF-710 and CRDF-818 with a series of precise experiments that will: 1. Elucidate the nature of the HLB signal(s) 2. Provide additional evidence on its transmission in terms of movement across tissues and between trees though underground organs. 3. Determine the progression of physical symptoms from its inception. 4. Examine the in-tree variation in CLas titer. Research commenced addressing objective 4, due to natural timing of events. Fifteen trees were selected for having branches with at least 11 leaves. This number of leaves was needed for analysis of CLas transport in both directions. The middle leaf in all 15 trees was disc grafted with HLB material and placed in the greenhouse until the presence of HLB symptoms. Trees are currently under observation. Experiments for objective 2 have been initiated. Two trees (one healthy and one HLB+) were root grafted in three different locations and placed in special pots large enough to accommodate the 2 trees. The trees have been placed in a greenhouse and are currently under observation. Objective 3 is also underway. Five healthy Valencia trees were disc grafted with HLB material and sent to Gainesville for symptom progression using Narrow-band imaging under polarized illumination.
This proposal is aimed at following previous work in CRDF-710 and CRDF-818 with a series of precise experiments that will: 1. Elucidate the nature of the HLB signal(s) 2. Provide additional evidence on its transmission in terms of movement across tissues and between trees though underground organs. 3. Determine the progression of physical symptoms from its inception. 4. Examine the in-tree variation in CLas titer. Research commenced addressing objective 4, due to natural timing of events. Fifteen trees were selected for having branches with at least 11 leaves. This number of leaves was needed for analysis of CLas transport in both directions. The middle leaf of all 15 trees was disc grafted with HLB material and placed in the greenhouse until the presence of HLB symptoms. Experiments for objective 2 are in under way. Trees, both HLB-affected and healthy, have been selected and special pots purchased. For this experiments, a healthy and a HLB tree will be root grafted and placed in a larger, flexible pot until HLB symptoms appear.
The Genome of Candidatus Liberibacter asiaticus (CLas) revealed the presence of luxR that encodes LuxR protein, one of the two components typical of bacterial “quorum sensing” or cell-to-cell communication systems. Interestingly, the genome lacks the second components; luxI that produce Acyl-Homoserine Lactones (AHLs) suggesting that CLas has a solo LuxR system. In the current project, we will test the effect of AHL-producing citrus plants on the pathogencity of CLas. Although the postdoctoral researcher has joined our lab two months later than the proposed starting date, we have selected different Lux-I genes from different bacteria expressing different AHLs. The selected genes included TraI (accession number: L22207.1) from Agrobacterium tumefaciens, AhyI (accession number: CAA61653) from Aeromonas hydrophilia, AYsI (accession number: DQ058009) from Agrobacterium vitis, CepI (accession number: AF019654.1) from Burkholderia cepacia, CviI (accession number: AY277257.1) from Chromobacterium violaceum, EsaI (accession number: L32183) from Panteoa stewartii, ExpI (Accession number: AY507108.1) from Pectobacterium carotovorum, LasI (protein ID: AHW72185.1) from Pseudomonas aeruginosa, RhlI (accession number: U11811.1) from Pseudomonas aeruginosa, PhzI (accession number: AAC18898.1) from Pseudomonas fluorescens, CinI (accession number: AF210630) from Rhizobium leguminosarum bv. Viciae, RhiI (protein ID: CAK10388) from Rhizobium leguminosarum bv. Viciae, RaiI (accession number: AJ427969) from Rhizobium leguminosarum bv. Viciae, CerI (accession number: AF016298) from Rhodobacter sphaeroides, SwrI (accession number: U22823.1) from Serratia liquifaciens, SinI (Protein ID: CAC46418.1) from Sinorhizobium meliloti, VanI (accession number: U69677) from Vibrio anguillarum, LuxI (protein ID: AAD48474.1) from Vibrio fischeri, LuxM (accesion number: L13940) from Vibrio harveyi, Yenl (accession number: X76082.1) from Yersinia enterocolitica, YpsI (accession number: AF079973.1) from Yersinia pseudotuberculosis, and YtbI (accession number: AF079136.2) from Yersinia pseudotuberculosis. Currently, we are synthesizing the genes in order to insert them in CTV-based vector prior to the infiltration inside citrus trees.
Citrus trees transformed with a chimera AMP and a thionin alone showed remarkable resistance in citrus canker compared to control. These promising transgenic lines were replicated by grafting for HLB challenge. Replicated transgenic Carrizo lines expressing thionin, chimera and control were grafted with HLB infected rough lemon buds. Las titer was checked from new flush rough lemon leaves at six month after grafting. Las titer from 18.6-36.5 was detected in 90% of transgenics expressing the chimera. Some transgenic lines expressing thonin had the lower Las tilter(most in 33.3-36.4 ranges). Transgenic root sample were further tested and most were detected with las titer from 30 to 35. All root samples will be checked at 9 month for Las titer. Two new chimeral peptides (second generation) were developed and used to produce many Carrizo plants and Hamlin shoots. Transgenic carrizo plants carrying second generation AMPs were transferred to soil cones. DNA was isolated from 46 plants and 40 of them are PCR positive. To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was used to transform citrus. Flagellins are frequently PAMPS (pathogenesis associated molecular patterns) in disease systems and CLas has a full flagellin gene despite having no flagella detected to date. The consensus FLS2 clone was obtained and used to transform Hamlin and Carrizo so that resistance transduction may be enhanced in citrus for HLB and other diseases. Reactive Oxygen Species (ROS) assay showed typical ROS reaction in transgenic Hamlin indicating nbFLS is functional in citrus PAMP-triggered immunity. Trees showed significant canker resistance to spray inoculation. Replicated Carrizo and Hamlin were challenged with ACP feeding. Las titer will be tested periodically. To disrupt HLB development by manipulating Las pathogenesis, a luxI homolog potentially producing a ligand to bind LuxR in Las was cloned into binary vector and transformed citrus. Both transformed Carrizo and Hamlin were obtained. Replicated transgenic Carrizo plants were challenged by ACP feeding. Las tilter will be tested soon. Transgenic Hamlin were propagated by grafting for HLB challenge. In collaboration with Bill Belknap two new citrus-derived promoters have been tested using a GUS reporter gene and have been shown to have extraordinarily high levels of tissue-specific expression. The phloem-specific promoter was used to create a construct for highly phloem specific expression of the chimeral peptide using citrus genes only. A Las expressed gene with a nuclear-localization sequence has been identified and studied, including creating transgenic citrus that express this p235 gene. Carrizo transformed with this gene displays leaf yellowing similar to that seen in HLB-affected trees. Gene expression levels, determined by RT-qPCR amplification, correlated with HLB-like symptoms. P235 translational fusion with GFP shows the gene product binds to citrus chloroplasts. Antibodies (ScFv) to the Las invA and TolC genes, and constructs to overproduce them, were created by John Hartung under an earlier CRDF project. We have transgenic Carrizo reflecting almost 400 independent transgenic events and 17 different ScFv ready for testing. A series of AMP transgenics scions produced in the last several years continue to move forward in the testing pipeline. Many trees are in the field and some are growing well but are not immune to HLB. A large number of ubiquitin::D4E1 and WDV::D4E1 plants and smaller numbers with other AMPs are replicated and now in the field.
Objective 1: Assess canker resistance conferred by the PAMP receptors EFR and XA21 Three constructs were used for genetic transformation of Duncan grapefruit and sweet orange as part of a previous grant: EFR, EFR coexpressed with XA21, and EFR coexpressed with an XA21:EFR chimera. Putative transgenics are currently being verified by PCR in the Jones lab, and three PCR positive plants have been identified so far. To ensure that there will be sufficient events to analyze to come to a conclusion about the effectiveness of these genes, we will initiate more transformations in Duncan grapefruit at the Core Citrus Transformation Facility at UF Lake Alfred. Objective 2: Introduction of the pepper Bs2 disease resistance gene into citrus Constructs are being created in the Staskawicz lab to express Bs2 under the 35S promoter and under a resistance gene promoter from tomato. Objective 3: Development of genome editing technologies (Cas9/CRISPR) for citrus improvement The initial target for gene editing is the citrus homolog of Bs5 of pepper. The recessive bs5 resistance allele contains a deletion of two conserved leucines. The citrus Bs5 homolog was sequenced from both Carrizo citrange and Duncan grapefruit, and conserved CRISPR targets were identified. Three CRISPR constructs are being created in the Staskawicz lab: 1) A construct targeting two sites that will produce a deletion in Bs5 in both Carrizo and Duncan (the bs5 transgene will be added); 2) A construct targeting a site overlapping the two conserved leucines, containing a bs5 repair template for Carrizo that will not be cut; and 3) a construct targeting the same site, with a repair template for Duncan grapefruit.
Chimeral constructs that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab) are being tested and are among the most promising transgenics we have created, along with thionin transgenics. Trees transformed with a chimera AMP showed remarkable resistance in citrus canker compared to control. These promising transgenic lines were replicated by grafting for HLB challenge. Transgenic Hamlin lines expressing thionin were grafted onto Carrizo for HLB challenge. Replicated transgenic Transgenic Carrizo lines expressing thionin, chimera and control were grafted with HLB infected rough lemon. Promising resistance to HLB was observed based on plant growth and phenotype. Las titer is being checked from root and new flush rough lemon leaves. Two new chimeral peptides from citrus genes only were developed and used to produce many Carrizo plants and Hamlin shoots which will be tested soon as part of the next generation of this project. To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was used to transform citrus. Flagellins are frequently PAMPS (pathogenesis associated molecular patterns) in disease systems and CLas has a full flagellin gene despite having no flagella detected to date. The consensus FLS2 clone was obtained and used to transform Hamlin and Carrizo so that resistance transduction may be enhanced in citrus for HLB and other diseases. Reactive Oxygen Species (ROS) assay showed typical ROS reaction in transgenic Hamlin indicating nbFLS is functional in citrus PAMP-triggered immunity. Trees showed significant canker resistance to spray inoculation. To disrupt HLB development by manipulating Las pathogenesis, a luxI homolog potentially producing a ligand to bind LuxR in Las was cloned into binary vector and transformed citrus. Both transformed Carrizo and Hamlin were obtained. Further investigation are underway. In collaboration with Bill Belknap two new citrus-derived promoters have been tested using a GUS reporter gene and have been shown to have extraordinarily high levels of tissue-specific expression. The phloem-specific promoter was used to create a construct for highly phloem specific expression of the chimeral peptide using citrus genes only. Transgenic plants of PP-2 hairpins (for suppression of PP-2 through RNAi to test possible reduction in vascular blockage even when CLas is present) and of PP-2 directly are grafted in the greenhouse. 40 putative transgenic plants transformed with citGRP1 were tested by PCR and twenty two of them were confirmed with citGRP1 insertion. RNA was isolated from some and RT-PCR showed gene expression. Some transgenics with over-expression of citGRP1 had increased resistance to canker by detached leaf assay but do not appear as potent as some other AMPs. Transgenic Carrizo and Hamlin with peach dormancy genes show no evidence of enhanced or accelerated dormancy A Las expressed gene with a nuclear-localization sequence has been identified and studied, including creating transgenic citrus that express this p235 gene. Carrizo transformed with this gene displays leaf yellowing similar to that seen in HLB-affected trees. Gene expression levels, determined by RT-qPCR amplification, correlated with HLB-like symptoms. P235 translational fusion with GFP shows the gene product binds to citrus chloroplasts. Antibodies (ScFv) to the Las invA and TolC genes, and constructs to overproduce them, were created by John Hartung under an earlier CRDF project. We have transgenic Carrizo reflecting almost 400 independent transgenic events and 17 different ScFv ready for testing. A series of AMP transgenics scions produced in the last several years continue to move forward in the testing pipeline. Many trees are in the field and some are growing well but are not immune to HLB. A large number of ubiquitin::D4E1 and WDV::D4E1 plants and smaller numbers with other AMPs are replicated and now in the field.
In the July-September 2015 period of our work, we have continued to finish strain constructions and have begun Affymetrix GeneChip experiments in order to examine expression of transcripts in host Sinorhizobium meliloti due to introduced Candidatus Liberibacter asiaticus (CLas) transcription factor genes. Previously we showed that the cloned CLas rpoH gene can complement phenotypes of an S. meliloti double rpoH1 rpoH2 deletion mutant. We have now isolated total RNA from the S. meliloti rpoH1H2 mutant strain expressing CLas rpoH and prepared labeled cDNA. Controls for this experiment are: S. meliloti rpoH1H2 carrying the empty vector, pSRK-Gm; and S. meliloti rpoH1H2 carrying S. meliloti rpoH1, cloned in pSRK-Gm. All samples (3 biological replicates of each of 3 strains) have been submitted to our campus facility for hybridization to the S. meliloti genome chip, which will reveal the total transcriptome arising from CLas rpoH expression. Beyond rpoH, we intend to study 6 transcription factors from CLas . As described in the previous report, we are creating S. meliloti host strains with deletions for each of the S. meliloti genes corresponding to those CLas regulators, so we can optimally measure function of the introduced CLas genes. As of July report, we had constructed the visRN and ldtR mutants. We have since constructed an lsrB mutant. The S. meliloti lsrB deletion strain grows poorly. Using the cloned CLas lsrB gene carried on plasmid pSRK-Gm, we showed that induced expression of CLas lsrB partly rescues the poor growth phenotype of the S. meliloti mutant. We are encouraged that this shows at least partial function of CLas LsrB in S. meliloti. We are working on construction of a double phrR1 and phrR2 mutant; meanwhile we have introduced the cloned CLas phrR gene into wild type S. meliloti, in case we are unable to make the phrR1 phrR2 double mutant.. Construction of a ctrA deletion strain requires an extra step, since ctrA is essential for S. meliloti viability. Our progress to date includes successful cloning of the CLas and S. meliloti ctrA genes in vector pSRK-Gm. Each of these plasmids was introduced into S. meliloti containing a single crossover of the ctrA deletion construct. In parallel, we have constructed wild type S. meliloti strains containing the cloned CLas and Sm ctrA genes. We will screen these two strains (overexpressing either CLas ctrA or S. meliloti ctrA) in parallel on sucrose. If the optimized CLas ctrA gene does not allow for viability of a S. meliloti ctrA deletion strain, then we will study the CLas CtrA regulator in wild type S. meliloti.
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