Our project aims to provide durable long term resistance to Diaprepes using a plant based insecticidal transgene approach. In this quarter, all transgenic lines as described in the project proposal have been regenerated and most plants have been acclimatized and transferred into the greenhouse for further growth. A few putative transgenic lines have been transferred to fresh in vitro rooting medium in efforts to stimulate root production. Root samples from 21 lines have been analyzed for gene expression using qPCR. Of them, 12 were determined to be high expressers while the rest were medium to low in expression. We are in the process of evaluating the remaining greenhouse acclimated lines for gene expression. The high expresser lines will be propagated for subsequent evaluation with Diaprepes neonates.
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. Seven transgenics have survived and passed a PCR screen, and these have been grafted onto rootstocks. Grafted plants are currently growing, and will be tested for responsiveness to the elf18 ligand for EFR and for canker resistance. To ensure that there will be sufficient events to analyze to come to a conclusion about the effectiveness of these genes, we have initiated more transformations in Duncan grapefruit at the Core Citrus Transformation Facility at UF Lake Alfred. In addition, we have added the recently-identified Cold Shock Protein Receptor (CSPR) to the transformation queue. Selection is underway, but the GFP marker is not expressed in citrus, and the protocol is being optimized. Objective 2: Introduction of the pepper Bs2 disease resistance gene into citrus Two constructs were created to co-express Bs2 with other R genes that may serve as accessory factors for Bs2. These constructs were provided to the Lake Alfred transformation facility, but the transformation attempts have so far been unsuccessful, possibly due to negative effects of the constructs in Agrobacterium or in citrus. Troubleshooting of these transformations with a negative control construct is underway. 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 homologs were sequenced from both Carrizo citrange and Duncan grapefruit, and conserved CRISPR targets were identified. For proof of concept, we are targeting mutating the native citrus Bs5 alleles while simultaneously replacing the gene with the effective resistance allele. Two editing constructs have been created, one targeting a site overlapping the two conserved leucines, and one targeting two flanking sites to create a deletion in Bs5. Both constructs have been verified to function by co-delivery into Nicotiana benthamiana leaves with another construct carrying the targeted DNA from Carrizo or Duncan varieties. These constructs have been prioritized for transformation into Carrizo citrange, and transformations are underway at UC Davis. Transformants with mutations in Bs5 that contain the replacement bs5 allele will be selected and tested for canker resistance.
The project has three objectives: (1) Confirm HLB resistance/tolerance in transgenic citrus lines. (2) Determine the chimerism of the HLB-resistant/tolerant transgenic lines. (3) Confirm HLB resistance in citrus putative mutants (nontransgenic lines). For objective 1, we are propagating a number of citrus transgenic lines overexpressing Arabidopsis defense genes. Our previous results indicated that these transgenic lines are likely resistant or highly tolerant to HLB. The progeny plants are growing in the greenhouse. For objective 2, we performed on round of real-time quantitative PCR (qPCR) to determine the chimerism of the HLB-resitant/tolerant transgenic lines. The results showed some of the lines may be chimeric. We are repeating the qPCR experiment. For objective 3, we are propagating the previously generated gamma ray-mutagenized mutant lines that are likely resistant/tolerant to HLB. The progeny plants are growing in the greenhouse.
During the last quarter of 2015 Core Citrus Transformation Facility (CCTF) continued to operate as planned and produced transgenic Citrus plants. The number of orders received for the last three months jumped to nine. Two of those orders were for transgenic Duncan grapefruit and the rest are the set of seven orders for production of genetically modified Pineapple orange. CCTF still has not received two groups of orders announced by prospective clients about 5-6 months ago. Also, one set of orders that included the use of three different vectors was withdrawn and the client is working on re-cloning of all vectors. CCTF produced 51 plants within last quarter. These plant belong to multitude of orders, altogether nine of them, some of which are old and some newer. Six of the produced plants were Valencia oranges, 11 were Duncan grapefruit, and the rest were Carrizo citrange. As I have previously reported to Dr. H. Browning, the analysis of transgenic plants carrying NPR1 gene was successfully completed by Yosvanis Acanda from Mature tissue transformation facility. Out of 67 tested transgenic rootstock plants produced by the CCTF, 23 were shown to be high expressers of NPR1. These plants are being kept in the CCTF s greenhouse until they reach the size at which they can be propagated so that they can serve as rootstocks for both transgenic and WT scion plants.
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 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.
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