HLB’s impacts have led to grower interest in advanced production and harvesting systems with the potential for early and sustainable yield, as well as ease of harvest and other management efficiencies. The goal of this project is to identify appropriate rootstocks among exiting field trials and those soon to be planted that are well suited to advanced citrus production and harvesting systems. Existing field trials previously planted with size-controlling rootstock candidates have continued to be observed, including the portion of the St. Helena project planted with dwarfing selections; a field day was held there in December to demonstrate the performance of the tree size controlling rootstocks. A 40-acre Hamlin/Valencia cooperative rootstock trial with trees planted between 300-500/acre has been monitored for tree growth and HLB responses, as was a high density planting of LB8-9 (Sugar Belle). Data are being compiled on rootstocks to identify new candidate rootstocks for larger’scale ACPS trials. Seedlings grown from previously untested Flying Dragon-derived hybrids, as well as from a range of other complex interspecific hybrids with tree size control potential, were grown to assess seedling vigor and other characteristics; selections were either discarded because of poor growth habits, excessive phenotypic variation, or poor germination. The best performers from this group were listed and substantial quantities of seed were harvested and made available to 3 different cooperators for new plantings and trials, hopefully to be planted in 2015 or spring 2016. Seeds were harvested from rootstock hybrids that are bearing their first fruit, and these have been planted in the greenhouse to assess germination rates, relative seedling vigor, and trueness to type. Additionally, large lots of seed from candidates for ACPS planting already identified from existing field trials were harvested and distributed to cooperating nurseries to produce trees for new trial opportunities in the 2015-16, with interested collaborative growers. Finally, a field day was also held to showcase some of the UFR rootstocks in a trial near Vero Beach, several of which are showing good performance even though they are affected by HLB, and with ACPS potential by virtue of smaller tree size combined with higher yield efficiencies.
The project has two objectives: (1) Increase citrus disease resistance by activating the NAD+-mediated defense-signaling pathway. (2) Engineer non-host resistance in citrus to control citrus canker and HLB. For objective 1, we continued optimizing the NAD+ treatment conditions. Both soil drench and foliar spraying of NAD+ were tested again. Foliar spraying did not provide significant protection against citrus canker, soil drench induced strong resistance against the pathogen. We previously observed strong systemic protection against canker by NAD+ one month after the treatment in upper new flushes and are repeating the experiment to confirm the systemic effects. We are also testing different recipes for stabilizing NAD+ on the surface of leaves or in soil, since this chemical is not stable under aqueous conditions. For objective 2, transgenic citrus plants expressing the Arabidopsis nonhost resistance genes are growing in greenhouse and will be tested for canker resistance. We are also propagating the transgenic plants to produce progenies for HLB resistance test. Citrus homologs of the Arabidopsis nonhost resistance genes have been cloned and sequenced. To test their functionality, we have cloned the citrus homologs into plant expression vector and have transformed into the corresponding Arabidopsis mutants. T1 plants are growing.
In this project, we have conducted transcriptome analysis on 25 samples of tolerant/susceptible citrus plants using RNA-Seq. We first performed bioinformatics analysis on RNA-seq data of three HLB-tolerant ‘Jackson’ grapefruit hybrid and three HLB-susceptible ‘Marsh’ grapefruit hybrid. We identified 686 differentially expressed (DE) genes between two groups using FDR threshold of 0.1. Among them, 247 genes were up-regulated and 439 were down-regulated in tolerant citrus trees. We have experimentally verified the expressions of 14 up-regulated genes and 20 down-regulated genes using real time PCR. 11 of 14 up-regulated genes and 18 of 20 down-regulated genes were validated. We performed Gene Ontology (GO) enrichment analysis of DE genes. Genes associated with beta-amyrin synthase, cycloartenol synthase and Camelliol C synthase were significantly up-regulated in the HLB tolerant citrus trees while terpene synthase genes (CiClev10014707, Ciclev10017785) were down-regulated in the tolerant citrus trees. Some PR-protein genes were significantly up-regulated in the tolerant citrus trees, including several TIR-NBS-LRR genes. Many cell wall degradation-related genes, such as cellulose synthase/transferase, cellulase and expansins were up-regulated in the susceptible citrus trees. Some glucan hydrolase genes were also up-regulated in the tolerant citrus trees. These genes may play important roles in symptom development. The DE genes were also enriched in two classes of RLKs, LRR-RLKs and DUF26-RLKs. We then applied the MapMan software to identify DE genes related to disease response. The MapMan annotated 155 DE genes related to disease response. We found multiple pathways have been suppressed or activated in HLB tolerant citrus tree, which lead to the promotion of the basal resistance or immunity in citrus tree. For examples, suppression of beta glucanases, DMR6-like genes, expansin and DET2 uphold the suppression of immunity of citrus tree and activation of NPR1-like genes also induces the immune responses to HLB. The mechanism how to trig the suppression and activation of these genes is still unclear. Further experimental studies on the NBS-LRR and RLK genes differentially expressed between HLB tolerant and HLB susceptible citrus trees may help understand the HLB related receptor genes. Meanwhile, our functional analysis also showed that the HLB development related genes were enriched in HLB susceptible citrus trees, such as viral life cycle and symbiosis related genes. We predicted a protein-protein interaction (PPI) network of citrus using the PPIs of Arabidopsis. There are 1259 proteins and 2298 interactions in our Citrus PPI network. Among 1259 proteins, 42 proteins are differentially expressed between the HLB resistance and susceptible citrus. An interested PPI sub-network includes 14 citrus NPR1-likes proteins and three TGA proteins. There were four NPR1-like genes were significantly up-regulated in HLB tolerant citrus trees and one NPR1-like gene up-regulated in HLB sensitive citrus trees. There is also one TGA gene up-regulated in HLB tolerant citrus trees. Another interested sub-network includes the RPS2 protein. There were two LRR kinase receptors significantly up-regulated in HLB tolerant citrus trees and four LRR kinase receptors significantly up-regulated in sensitive citrus trees. In conclusion, by profiling the transcriptome of HLB tolerant and HLB susceptible citrus trees, we found that multiple pathways have been suppressed or activated in HLB tolerant citrus tree, which lead to the promotion of the basal resistance or immunity of citrus trees. This study may help us understand HLB-tolerance and provide guidance for breeding HLB-tolerant citrus in the future. The down-regulation genes can be served as the candidate targets for RNAi to improve HLB tolerance in citrus trees. Two manuscripts were prepared: one published, and the other has been submitted.
In this project, we have conducted transcriptome analysis on 25 samples of tolerant/susceptible citrus plants using RNA-Seq. We first performed bioinformatics analysis on RNA-seq data of three HLB-tolerant ‘Jackson’ grapefruit hybrid and three HLB-susceptible ‘Marsh’ grapefruit hybrid. We identified 686 differentially expressed (DE) genes between two groups using FDR threshold of 0.1. Among them, 247 genes were up-regulated and 439 were down-regulated in tolerant citrus trees. We have experimentally verified the expressions of 14 up-regulated genes and 20 down-regulated genes using real time PCR. 11 of 14 up-regulated genes and 18 of 20 down-regulated genes were validated. We performed Gene Ontology (GO) enrichment analysis of DE genes. Genes associated with beta-amyrin synthase, cycloartenol synthase and Camelliol C synthase were significantly up-regulated in the HLB tolerant citrus trees while terpene synthase genes (CiClev10014707, Ciclev10017785) were down-regulated in the tolerant citrus trees. Some PR-protein genes were significantly up-regulated in the tolerant citrus trees, including several TIR-NBS-LRR genes. Many cell wall degradation-related genes, such as cellulose synthase/transferase, cellulase and expansins were up-regulated in the susceptible citrus trees. Some glucan hydrolase genes were also up-regulated in the tolerant citrus trees. These genes may play important roles in symptom development. The DE genes were also enriched in two classes of RLKs, LRR-RLKs and DUF26-RLKs. We then applied the MapMan software to identify DE genes related to disease response. The MapMan annotated 155 DE genes related to disease response. We found multiple pathways have been suppressed or activated in HLB tolerant citrus tree, which lead to the promotion of the basal resistance or immunity in citrus tree. For examples, suppression of beta glucanases, DMR6-like genes, expansin and DET2 uphold the suppression of immunity of citrus tree and activation of NPR1-like genes also induces the immune responses to HLB. The mechanism how to trig the suppression and activation of these genes is still unclear. Further experimental studies on the NBS-LRR and RLK genes differentially expressed between HLB tolerant and HLB susceptible citrus trees may help understand the HLB related receptor genes (PLoS ONE 10(3): e0121893). Meanwhile, our functional analysis also showed that the HLB development related genes were enriched in HLB susceptible citrus trees, such as viral life cycle and symbiosis related genes. We predicted a protein-protein interaction (PPI) network of citrus using the PPIs of Arabidopsis. There are 1259 proteins and 2298 interactions in our Citrus PPI network. Among 1259 proteins, 42 proteins are differentially expressed between the HLB resistance and susceptible citrus. An interested PPI sub-network includes 14 citrus NPR1-likes proteins and three TGA proteins. There were four NPR1-like genes were significantly up-regulated in HLB tolerant citrus trees and one NPR1-like gene up-regulated in HLB sensitive citrus trees. There is also one TGA gene up-regulated in HLB tolerant citrus trees. Another interested sub-network includes the RPS2 protein. There were two LRR kinase receptors significantly up-regulated in HLB tolerant citrus trees and four LRR kinase receptors significantly up-regulated in sensitive citrus trees. In conclusion, by profiling the transcriptome of HLB tolerant and HLB susceptible citrus trees, we found that multiple pathways have been suppressed or activated in HLB tolerant citrus tree, which lead to the promotion of the basal resistance or immunity of citrus trees. This study may help us understand HLB-tolerance and provide guidance for breeding HLB-tolerant citrus in the future. The down-regulation genes can be served as the candidate targets for RNAi to improve HLB tolerance in citrus trees.
Analysis and writing continue in preparation for the end of this project in June. One student is completing her MS degree, and with her data completed and analyzed, manuscripts are being developed. These may not be completed by the termination date of the project, but we hope to have at least first drafts done. The FT3 gene is also being put into the CTV vector and is being put into CRISPR/Cas systems in collaborations with others.
A chimeral construct that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab) is being tested. Many transformed Carrizo with the chimera AMP were obtained. Exposure to canker inoculum showed remarkable resistance in chimera compared to control. RNA was isolated from 16 transgenic Hamlin containing Chimera. RT-qPCR showed 50% of them have relative high gene expression. One of them showed over hundred times higher expression compare to plant expressing the lowest level of chimera. These promising transgenic lines were replicated by grafting for HLB challenge. About 30 Hamlin transformed with thionin also were obtained. Twenty transgenic lines were confirmed containing thionin gene by PCR. RNA was isolated from 16 transgenic Hamlin containing thionin. Six of them have relative high gene expression by RT-qPCR. These transgenic lines will be replicated for HLB challenge. Two new chimeral peptide have been developed and is used to transform citrus. Many transformed Carrizo shoots with new chimera construct were obtained. Some were transferred to the rooting medium. Replicated trangenic lines expressing chimera, thionin and D4E1were grafted with HLB infected rough lemon. Las tilter will be checked by qPCR periodically. To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was cloned into pBinARSplus vector 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. Many putative transformants were generated on the selective media. DNA was isolated from 80 of them: 38 Carrizo and 7 Hamlin are positive by PCR test. Reactive Oxygen Species (ROS) assay showed typical ROS reaction in three of transgenic Hamlin which suggest nbFLS is functional in citrus PAMP-triggered immunity. However, there is only slight canker resistance by infiltration test. Spray inoculation was tried and some of them show obvious canker resistance. To confirm that high ROS production was not due to variability in Hamlin, we examined 40 Hamlin seedlings and no or very low level ROS production was detected. In contrast, relatively higher ROS production was detected from wild-type Carrizo seedings compared to Hamlin seedlings. Two potential FLS2 orthologues were identified in Hamlin and their expression was shown much lower compare to nbFLS2. Replicated trangenic Carrizo lines expressing nbFLS2 were challenge with ACP. Las titer will be checked by qPCR 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. Further investigation are underway. A series of transgenics scions produced in the last several years continue to move forward in the testing pipeline. Several D35S::D4E1 sweet oranges show initial growth in the field which exceeds that of controls. A large number of ubiquitin::D4E1 and WDV::D4E1 plants and smaller numbers with other AMPs are replicated and in early stages of testing. 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 is being used to create a construct for highly phloem specific expression of the chimeral peptide using citrus genes only.
Trees of seemingly HLB resistant/tolerant sweet orange-like hybrids and mandarin -types have been propagated on x639. Replicated trials with standards will be established. Six locations each of all sweet orange-like together and 4 with all mandarins will be established with 6-8 trees of each cultivar at each site. We have identified cooperators (in Ridge, IR and Gulf coast) for complete replicated block plantings at each site. In October 2013, 34 unique genotypes (USDA hybrids) some of which appear to have tolerance to HLB, and 16 standard commercial varieties were exposed to an ACP no-choice feeding trial and have been transferred to the field at Ft. Pierce Fl. Standard growth measurements and disease ratings were initiated in July 2014 and will continue on a monthly basis. As of December 2014, the first HLB symptoms are apparent. Evaluation of existing standard and non-standard cultivars (‘Hamlin’, ‘Temple’, ‘Fallglo’, ‘Sugar Belle’, ‘Tango’, and ‘Ruby Red’) for HLB resistance/tolerance is complete. In August 2010, the plants were established at Pico’s farm in Ft. Pierce Fl. Data on the growth rate, disease severity, and Candidatus Liberibacter asiaticus (CLas) titer levels have been collected since April 2012. During the 4-year period, there were significant differences in disease severity, stem diameter, and CLas levels among the varieties. All trees exhibited symptoms of HLB and tested positive for CLas, with similar titers measured at most recent sample dates.’Fallglo’ had the lowest incidence of HLB symptoms, whereas ‘Ruby Red’ had the highest incidence. ‘Ruby Red’ also appears to be in significant decline. Despite the high initial titer levels found in ‘SugarBelle’, it had the greatest overall increase in diameter and was the healthiest in overall appearance. In Nov. 2014 ‘Temple’ trees had significantly greater fruitload, with 26 fruit/tree, followed by ‘Tango’ with 10 fruit /tree, ‘Hamlin/Kinkoji’ with 5 fruit/tree and all others with 0-1.4 fruit/tree. Production was compromised in all varieties by the severe HLB pressure at this site, and commercial value of the observed tolerance remains uncertain. Progress has been made on the antibiotic treatment of HLB infected bud-wood to compare growth at different levels of CLas infection. Bud-wood of nine HLB symptomatic varieties, 3 fairly resistant (‘Temple’, GnarlyGlo’, and ‘Nova’) 3 tolerant (‘Jackson’, FF-5-51-2, and Ftp 6-17-48), and 3 susceptible (‘Flame’, ‘Valencia’, and ‘Murcott’). In November 2013 and May 2014, HLB positive bud-wood was treated with various concentrations of penicillin and streptomycin and grafted on sour orange rootstock. Standard growth measurements (stem diameter and height), disease severity were evaluated and leaves were sampled for qPCR analysis. Evaluations and sampling will continue on quarterly basis. Development of periclinal chimeras with resistant vascular tissue from Poncirus and remaining layers from sweet orange is currently underway. One hundred and fifty etiolated seedlings of the trifoliate ‘Rubidoux’ and the sweet orange ‘Hamlin’ have been approach grafted together. Generation of new chimeras has been difficult. Several adventitious buds have emerged from the treated graft region, with several appearing to be chimeral. The newly emerged plants will be tested using LC/MS to determine the origin of the three layers. To increase the success rate, additional plants will be grafted over the next twelve months. A method for the rapid identification of potential sources of HLB resistance is also being developed. This project involves the screening of citrus seedlings at the 3 to 5 leaf stage, or very small micrografted trees, that are exposed to HLB infect ACP feeding. CLas titer levels, using real time PCR, are evaluated at 3, 6, and 9 weeks Seedlings of Hamlin and Dancy show early CLas proliferation and systemic movement. Only very low levels of CLas have been observed in Carrizo.
A transgenic test site at the USDA/ARS USHRL Picos Farm in Ft. Pierce supports HLB/ACP/Citrus Canker resistance screening for the citrus research community. There are numerous experiments in place at this site where HLB, ACP, and citrus canker are widespread. The first trees have been in place for over five years. A number of successes have already been documented at the Picos Test Site funded through the CRDF. The UF Grosser transgenic effort has identified promising material, eliminated failures, continues to replant with new advanced material, with ~200 new trees in April 2015 (Grosser, personal comm.). The ARS Stover transgenic program has trees from many constructs at the test site and is seeing some modest differences so far, but new material is being planted this spring that has shown great promise in the greenhouse (unpublished). A trial of more than 85 seedling populations from accessions of Citrus and citrus relatives (provided as seeds from the US National Clonal Germplasm Repository in Riverside, CA) has been underway for 5 years in the Picos Test Site. P. trifoliata, Microcitrus, and Eremocitrus are among the few genotypes in the citrus gene pool that continue to show substantial resistance to HLB (Lee et al., in preparation), and P. trifoliata also displayed reduced colonization by ACP (Westbrook et al., 2011). A new UF-Gmitter led association mapping study has just been initiated using the same planting, to identify genes associated with HLB- and ACP-resistance. A collaboration between UF, UCRiverside and ARS is well-underway with more than 1000 Poncirus-hybrid trees (including 100 citranges replicated) being evaluated to map genes for HLB/ACP resistance. Marked differences in initial HLB symptoms and Las titer were presented at the 2015 International HLB conference (Gmitter et al., unpublished) and David Hall is now assessing ACP colonization. Several USDA citrus hybrids/genotypes with Poncirus in the pedigree have fruit that approach commercial quality, were planted within the citrange site. As of April 2014 at the Picos Test Site, several of these USDA hybrids had grown to a height of seven ft, with dense canopies and good fruit set, while sweet oranges are stunted (3 ft) with very low vigor (Stover et al., unpublished). A Fairchild x Fortune mapping population will be planted at the Picos Test Site this spring in an effort led by Mike Roose to identify genes associated with tolerance. This replicated planting will also include a number of related hybrids (among them our easy peeling remarkably HLB-tolerant 5-51-2) and released cultivars. Valencia on UF Grosser tertazyg rootstocks have been at the Picos Test Site for several years, having been Las-inoculated before planting, and several continue to show excellent growth compared to standard controls (Grosser, personal comm.).
We continue to conduct the weekly Agrobacterium-mediated transformations and screen putatively transformed mature scion and rootstock shoots for clients. The total number of transgenics produced and detected thus far (excluding the 157 reported earlier) is ~100, and ~ half of these survived primary and secondary grafting (excluding the 66 reported earlier). The transformation efficiency for a plasmid with no reporter gene and a weak promoter driving the npt11 selectable marker was 2.8% for mature scion and rootstock. As expected, mature rootstock gave a higher transformation efficiency than mature scion. As previously mentioned, the weaknesses of this protocol are the high number of escapes particularly with constructs having weak promoters driving the npt11 selectable marker, difficulties with micro-grafting, and for certain constructs with no reporter genes, the difficulties in screening by PCR. PCR is expensive, time-consuming, labor intensive and prone to error. After the standard tissue culture protocol, growth in selective liquid media has assisted in identifying 4 transgenic events, which indicates that better selection is important. GFP expressing transgenic shoots were regenerated from mature citrus after biolistics, but subsequently died after micro-grafting. We will consider purchasing mature scion from nurseries solely for biolistics because we cannot produce enough in the growth room to facilitate the weekly Agrobacterium transformations and optimize biolistics. This mature scion will not enter the growth facility unless it has been cleaned through shoot-tip grafting. Parameters still to be optimized include use of the hepta adapter, the number of bombardments per treatment, growth stages (days after culture initiation) of target explants, and osmotic treatment on medium containing sorbitol and mannitol (hours and concentrations). We have had to find an alternate California seed source because certain varieties that we regularly use in tissue culture are contaminated by a fungus. Spraying fungicide does not eliminate this seed borne fungus. A manuscript entitled, “Genetic Transformation of Commercially Important Mature Citrus Scions” authored by Hao Wu, Yosvanis Acanda, Alka Shankar, Michael Peeples, Calvin Hubbard, Vladimir Orbovi. and Janice Zale has been accepted for publication in Crop Science.
Objective 1: Generate functional EFR variants (EFR+) recognizing both elf18-Xac and elf18-CLas A number of strategies to engineer an EFR variant that recognized elf18-Clas were tested over the grant period, but none were successful. These strategies included PCR mutagenesis of EFR, screening of natural variants in an extensive Arabidopsis accession collection, creating targeted mutations based on the modeled interactions among elf18, EFR, and BAK1, and testing high-throughput selection strategies such as phage display and fluorescence activated cell sorting. Funding for this activity has ended and effort toward this objective has ceased. Objective 2. Generate functional XA21-EFR chimera (XA21-EFRchim) recognizing axYS22-Xac. This objective was completed and a manuscript describing the XA21-EFR chimera and the complementary EFR-XA21 chimera has been published (Holton et al., 2015, PLoS Pathogens 11, e1004602). Objective 3: Generate transgenic citrus plants expressing both EFR+ and XA21-EFRchim. Putative transgenic citrus plants have been generated for four constructs: EFR, EFR plus XA21, EFR plus XA21-EFRchim, and the empty vector pCAMBIA2201. To date, 174 Duncan grapefruit, 20 sweet orange, and 5 Carrizo citrange plants have been tranferred to soil, and PCR analysis is beginning to determine whether the intact transgenes are present.
The interest for transgenic Citrus material remains strong which keeps Core Citrus Transformation Facility (CCTF) busy at all times. This interest is exemplified by the fact that eight more orders were received in the last three months. Due to the availability of appropriate starting material needed for these orders, the work on some of them started almost immediately. CCTF also continued to service the old orders. Within this period, CCTF performed tasks associated with the production of transgenic plants within the lab and also took care of plants from different orders growing in the greenhouse. In the last three months more than 80 plants were produced and are result of work on 12 different orders (NPR1, NPR1-G, ELP3-G, ELP4-G, PR-2, MG113, MG36, X7-2, HGJ31, HGJ32, HGJ33, and HGJ34). These plants belonged to only two cultivars: Carrizo and Duncan. Most recent group of orders had to do with the ‘proof-of-concept’ experiments and the T-DNA in binary vectors contained GFP gene which reflected positively on the productivity of CCTF in this quarter. Another factor affecting productivity was that involvement of EM lab staff resulted in definition of methodology for detection of fluorescence in transgenic plants that belong to two orders. CCTF temporarily lost contact with the grower who supplied Duncan grapefruit as a source of seeds and as a result planting for new orders is not going as planned. Intense effort is under way to find another source of Duncan fruit. Initial contact was established with the employee at the Southern Gardens farm to get supply of Valencia seeds. Shipment of Carrizo seeds was received from nursery in California. Five more plants were produced for the CRDF order bringing total to more than 50. In the early phases of this project, there were regular bi-weekly meetings between Jackie Burns, and managers of both transformation facilities to discuss the on-going activities. In that period, rate of production of transgenic plants was discussed as well as choice of cultivars. There was a general understanding that there would be more transgenic rootstock cultivars produced by juvenile method than scion cultivars produced from mature tissue. Within the first 7-8 months of the project that is exactly what happened. Although initial projection was that first group of most promising transgenic rootstock plants would be propagated already, they were not. The first reason is that plants did not grow well this winter. The second reason is that according to previous agreement only a highest expressors of NPR1 should be multiplied but those plants have not been selected yet. Considering early recognition of higher rate of production of transgenic plants in CCTF, it was presumed that the work on search for high NPR1 expressors (both rootstocks and scions) would be done by mature tissue lab. This type of work involves isolation of RNA and the use of RT-PCR machine, and the members of the mature lab may be better skilled for this type of work than employees in the CCTF. Due to personnel structure in CCTF, there are some limitations on what can be accomplished there at the moment. Manager of mature tissue lab was told that her employees could use RT-PCR machine that is available in CCTF at any time. Consultations are continuing on where this work will be done. Recently, information was requested from the CCTF manager about what space would be needed (in square feet) for propagation of rootstock plants with NPR1 gene. Once selection of plants that are high NPR1 expressors is complete such information will be available.
Huanglongbing (HLB) is the most serious threat to the U.S. citrus industry. Although no known HLB-resistant citrus plants have been identified, some citrus relatives are substantially more tolerant, such as Citrus jambhiri (rough lemon) and Poncirus trifoliata. Genome analysis will improve our understanding of the HLB tolerance mechanisms. Genomic DNA from Citrus jambhiri was used to generate more than 235 M reads (2 X 100) representing about 34-fold physical coverage of the rough lemon genome. A reference-guided method was used to assemble the rough lemon genome. Compared with the haploid Clementine reference genome sequence, there were more than 2.5 million single-base differences and about 0.7 million insertion/deletion polymorphisms. The effect of these SNPs were annotated based on their position. Most of the identified SNPs were located outside of the known gene. RNA-sequencing data are being used for gene annotation. The genomes of Poncirus trifoliata and Citrus clementina, along with rough lemon, are being utilized to design the Agilent’s SureSelect probes, which will be used for target enrichment. Leaf samples of a diverse group of citrus accessions were collected, including mandarin, sweet orange, pumelo, rough lemon, and Poncirus. The methods for high throughput nuclear DNA extraction method is being tested to extract high-quality DNA for target enrichment and next generation sequencing. Poncirus trifoliata genomic sequence reads that match the nucleotide-site leucine-rich repeat (NBS-LRR) class disease resistance genes of Citrus clementina were obtained. These Poncirus sequence reads will be assembled to identify NBS-LRR genes those are homologous to C.clementina and C.sinensis. Aligned NBS-LRR gene sequences of Poncirus will be used as resistant resource pool to identify sequence variations associated with HLB disease.
We have concluded the activities of objective 1 of our proposal which have focused around finding a native citrus protein replacement for cecropin B the C-terminal component of the chimeric antimicrobial (CAP) protein. We had identified CsHAT52 using one set of bioinformatics tools and confirmed antimicrobial activity with a portion of this protein that we designated CsHAT22. Bioassay of CsHAT22 revealed a minimum inhibitory concentration (MIC) of 50 uM with Xanthomonas, 100 uM with Xylella and 300 uM with Liberibacteria crescens (Lc). Using two additional bioinformatics programs, PAGAL and SCAPEL and have successfully identified and tested 2 additional proteins, CsPPC20 and CsCHITI25 that were compared to CB and the N-terminal 21 amino acids of CB designated CBNT-21. Among the test strains used Xanthomonas was most susceptible to the peptides with CB and CBNT21 showing and MIC values 25 uM and the MIC values for CsPPC20 and CsCHITI25 were 50uM and 100uM respectively. Both Xylella and the BT-1 strain of Lc gave MIC values of 200 uM for CBNT21 against both Xylella and Lc BT-1. CsPPC20 was more active than BNT21 against Xylella giving an MIC value of150 uM and as active against Lc BT-1 giving an MIC value of 200uM. CsCHITI25 was as active as CsPPC20 against Xylella but not as active against Lc. Based on these results we have included CsPPC20 as an additional construct for testing in planta and excluded CHITI25. CTV vectors for expressing CsP14a, CsP14a-CB and CsP14a-CsHAT52 have been constructed and evaluation and testing of the efficacy of these vectors is underway. The construction of a CTV vector to express CsP14a-CsPPC20 is underway. Binary vectors for Agrobacterium-mediated transformation of CsP14a, CsP14a-CB, CsP14a-CsHAT52 and CsP14a-CsPPC20 have been completed and the transformation process of tobacco and Charrizo tissues is underway for the isolation of transgenic plants. This is being done at the plant transformation facility at UCDavis. We obtained transgenic Carrizo citrus transformed with NE-CB as a positive control. These plants are in the CRF and last week they were exposed to infected psyllids. Once transgenic Carrizo are obtained with the 4 vectors they will be transferred to the CRF for testing with infected psyllids for resistance against HLB.
Huanglongbing (HLB) is the most serious threat to the U.S. citrus industry. Although no known HLB-resistant citrus plants have been identified, some citrus relatives are substantially more tolerant, such as Citrus jambhiri (rough lemon) and Poncirus trifoliata. Genome analysis will improve our understanding of the HLB tolerance mechanisms. Genomic DNA from Citrus jambhiri was used to generate more than 235 M reads (2 X 100) representing about 34-fold physical coverage of the rough lemon genome. A reference-guided method was used to assemble the rough lemon genome. Compared with the haploid Clementine reference genome sequence, there were more than 2.5 million single-base differences and about 0.7 million insertion/deletion polymorphisms. RNA-sequencing data are being used for gene annotation. The genomes of Poncirus trifoliata and Citrus clementina, along with rough lemon, are being utilized to design the Agilent’s SureSelect probes, which will be used for target enrichment.
Improving Consumer Acceptance: 1. In efforts to reduce juvenility in citrus, transgenic Carrizo citrange have been successfully produced expressing the clementine CFT3 gene. We have very small micrografted plants flowering in the greenhouse and these plants have been evaluated by PCR to confirm presence of the CFT3 gene. A few micrografted trees flowered immediately. 2. Following the successful demonstration of the inducible cre-lox gene system, the plant transformation vector has been modified to contain our NPR1 gene and Agrobacterium mediated citrus transformation is underway to incorporate this gene. 3. Transgenic plants containing our stacked transgenes are being clonally propagated for disease resistance evaluation and the first trees will be challenged for HLB resistance in spring 2015. 4. Plants in our Indoor RES structure have not flowered this year. It is possible greenhouse temperature may have played a role in the flowering process. We will attempt to keep the greenhouse unheated this fall in hopes of initiating flowering in spring 2015 Additional Resistance Gene candidates: 1. Transgenic plants containing antimicrobial gene LIMA-B were propagated for field challenge. 2. OLL-8 sweet orange and W. Murcott were transformed with the CtNH1 gene (NPR1-like), using our protoplast/GFP transformation system. Small colonies and embryos were regenerated from OLL-8 with GFP expression.
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