The objective of this project is to find poncirus hybrids that exist now that are sufficiently tolerant and of sufficient horticultural and juice quality to be used now for new planting in the presence of high levels of Huanglongbing (HLB) inoculum. We believe there is a good chance that there mature budwood exists with these properties that could be available immediately for new plantings. Although these trees are not likely to be equal in juice and horticultural qualities of the susceptible varieties of sweet oranges grown in Florida, with their tolerance to HLB they could be an acceptable crutch until better trees are developed. We surveyed the trees at the Whitney field station and found 5 lines that we thought could be acceptable for juice. Those have been propagated and are beginning to be tested for tolerance and horticultural properties.
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 three years. Dr. Jude Grosser of UF has provided 550 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser planted an additional 89 trees including preinoculated trees of sweet orange on a complex tetraploid rootstock that appeared to confer HLB resistance in an earlier test. Dr. Kim Bowman has planted several hundred rootstock genotypes transformed with the antimicrobial peptide D4E1. Texas A&M Anti-ACP transgenics produced by Erik Mirkov and expressing the snow-drop Lectin (to suppress ACP) have been planted along with 150 sweet orange transgenics from USDA expressing the garlic lectin. Eliezer Louzada of Texas A&M has permission to plant his transgenics on this site, which have altered Ca metabolism to target canker, HLB and other diseases. More than 120 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) have been planted in a replicated trial in collaboration with Fred Gmitter of UF and Mikeal Roose of UCRiverside. Plants are being monitored for CLas development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding. Dr. Roose has completed initial genotyping on a sample of the test material using a “genotyping by sequencing” approach. Additional plantings are welcome from the research community.
Evaluation of existing standard cultivars (‘Temple’, ‘Fallglo’, ‘Sunburst’, ‘Sugar Belle’, ‘Tango’, ‘Hamlin’, and ‘Ruby’) for HLB tolerance/resistance is underway . Trees were planted in 2010, using a randomized complete block design, at Picos Farm, Ft. Pierce, Fl. HLB symptom development and tree growth (diameter and height) are being monitored on a monthly basis. All of the cultivars in this trial exhibit symptoms of HLB and have tested positive for Candidatus Liberibacter asiaticus (CLas). Preliminary results indicate that there are a range of host responses with ‘Temple’ in the most tolerant group. A second project involves the treatment of various resistant/tolerant citrus accessions and susceptible standards with various concentrations of antibiotics to generate a range of CLas titer levels. In February 2013, budwood with various concentrations of CLas, derived from the antibiotic treated plants, will be evaluated for their potential to result in HLB symptoms in disease free material. The budded plants will be evaluated for growth and HLB symptoms development over a 2-year period. Temporal progression and systemic movement of the bacteria in the inoculated plants will be determined along with HLB symptom development, and growth of the plants.. Development of periclinal chimera using resistant geneotypes and standard varieties is in progress. In vitro shoots have been established from nodal and internodal explants excised from mature, certified disease free plants of Red Carrizo, Temple, Hamlin, and Valencia. After root formation, chimeras will be generated using a procedure developed by Ohtsu (1994). After successfully generating the chimeras with HLB resistant vascular system and good fruit using the previously mentioned cultivars, additional cultivars such as ‘Sweet Orange’ and grapefruit will be added to this study. An additional study has been added to the project. Screening and evaluating new scion materials is a lengthy process and require multiple testing locations. Due to the urgency to develop tolerant/resistant material, a shorter evaluation cycle procedure is being investigated. If this screening method is successfully, it may be useful to quickly identify new sources of resistance varieties that may enhance and improve citrus production in Florida.
Dr. Guixia Hao, who has extensive experience in plant transformation and molecular biology, began working on this project 9/23/2012. New constructs have been used to transform citrus scions including hairpins to suppress PP-2 through RNAi (to test possible reduction in vascular blockage even when CLas is present), a citrus promoter driving citrus defensins (designed by Bill Belknap of USDA/ARS, Albany, CA), and genes which may induce deciduousness in citrus. Numerous putative transformants are present on the selective media. A chimeral construct that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab) is finally completed and will be used in transformations next quarter. A series of transgenics scions produced in the last several years, continue to move forward in the testing pipeline.
Development continued on new rootstocks with outstanding attributes for Florida production, including tolerance to HLB. Yield, fruit quality, and tree size data were collected from ten rootstock trials with early ripening scions. Propagation continued to prepare trees for four more field trials to plant this year. Propagations of supersour selections were prepared for budding of trees for field trials, and another group was prepared for field planting. Cooperative work was continued with a commercial nursery to multiply 250 advanced supersour selections for placement of trees into cooperative field trials with growers at multiple locations. Work continued to assess supersour tolerance of CTV, Phytophthora, and Diaprepes using carefully controlled tests in the greenhouse and the field. Preparations were made for controlled testing of supersour selections for tolerance to high pH. Specialized testing of the supersour hybrids and concurrent field trials will effectively identify specific supersour selections that are equal or superior to sour orange in horticultural attributes and effects on fruit quality, as well as provide disease resistance or tolerance. Experiments were initiated to study the most important components of the tolerance to HLB exhibited by some citrus rootstocks. The final stage of a study of metabolic changes in HLB infected germplasm is being completed to supplement the gene expression study completed last year, including HLB susceptible and tolerant cultivars. Detailed evaluation of specific defense-related genes continued, including CtCDR1 and CtPDF2, identified by microarray as being responsive to HLB in tolerant rootstocks. Constructs are being built using this knowledge and that will allow the creation of new cultivars with increased HLB tolerance using only citrus origin genes. Knowledge gained about specific citrus resistance genes will also help guide crosses for the creation of conventional hybrids with improved HLB tolerance or resistance. A study to define the interaction of rootstock tolerance with scion tolerance/susceptibility is nearly complete, and is expected to be published by mid-year. Additional trees were propagated to examine the effect of rootstock tolerance to HLB in trees with the scaffold composed of the tolerant variety. Trees were also propagated for a field planting that will examine the same high grafting technique. Collaborative work continued to assess rootstock interaction with scion, nutrition, and management factors in determining tree tolerance to HLB. A manuscript was published on collaborative work demonstrating the association of particular small RNAs and nutrition, with HLB infection. Collaborative work continued on the relationship of small RNA to the HLB tolerance of selected citrus genotypes. Collaborative work began to compare the early response of trees infected by HLB to those infected by CTV, both phloem-limited pathogens. Selected citrus plant resistance genes were inserted by genetic transformation into outstanding rootstock and scion cultivars to develop new varieties with increased resistance to HLB. More than 300 new transgenic rootstock selections with potential resistance to HLB were produced, targeting increased expression of the citrus resistance genes CtNPR1, CtEDS1, CtMOD1, CtEDS5, CtPAD4, CtNDR1, or CtACD1. Twenty-five new transgenic rootstocks with selected antimicrobial genes were propagated and entered into a replicated greenhouse test with ACP inoculation to assess tolerance to HLB. A field trial continued with selected transgenic rootstocks to evaluate performance under natural field infection with HLB. The field trees are nearly 100% infected with HLB, but differ widely in the severity of symptoms and the effect of HLB on plant growth. Three presentations were made at the International Citrus Congress on research progress in the USDA rootstock program, including descriptions of new USDA citrus rootstocks, rootstocks for tree size control, and methods to test citrus selections for HLB resistance.
The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, has spread to citrus growing regions nearly worldwide and adults transmit phloem-limited bacteria (Candidatus Liberibacter spp.) that are putatively responsible for citrus greening disease (huanglongbing). Host plant resistance ultimately may provide the most effective, economical, environmentally safe, and sustainable method of control. In earlier experiments we identified genotypes of Poncirus trifoliata and xCitroncirus sp. (hybrids of P. trifoliata and another parent species) that were resistant to ACP. One mechanism we investigated to see whether it contributed to this resistance was plant hormones. We sprayed salicylic acid, methyl jasmonate, and abscisic acid, which are all common plant hormones, on susceptible citrus plants to test the influence on host choice, oviposition, development, and survival of ACP. Abscisic acid cut the life span of adult ACP in half compared to untreated control plants. The plant hormones had no other strong effects on ACP, but abscisic acid may play a minor role in preventing oviposition by ACP. We are currently doing a third replication to verify effects of plant hormones on oviposition. In addition, we are preparing the data for publication in a peer-reviewed journal. Another mechanism that may confer resistance to ACP is the structure of the scelerenchyma (a fibrous ring) surrounding the phloem. The scelerenchyma in non-citrus plant species was found to prevent feeding by herbivorous insects. We are collaborating with a Research Entomologist at our facility to test whether ACP choose different feeding positions on leaves of resistant P. trifoliate versus suceptible xCitroncirus sp. We completed one replication and found differences in feeding position. We will soon initiate a second replication to verify our results. If we verify differences in feeding position on resistant and susceptible leaves, our collaborator will then evaluate the salivary sheaths from ACP and the scelerenchyma in leaves using scanning electron microscopy. Some of the research that we discussed in earlier progress reports is now in review or already published in peer-reviewed journals, including a paper on host plant resistance in HortScience, one in the journal Crop Science, a review paper in Entomologia Experimentalis et Applicata that focuses on control strategies of ACP, and a paper on miticides (investigating which miticides could be sprayed on plants and experimental ACP without killing them). Collaborators at the Fujian Academy of Agricultural Sciences conducted free-choice tests with all major groups of citrus and found differences among and within groups. Most groups of citrus were colonized by ACP, but lemons were the most preferred group and sour oranges and kumquats were the least preferred. The differences among citrus varieties within a group may be useful because volatile and phloem contents that differ between the least and most preferred species can be compared. FAAS continues their screening of germplasm including CRC accessions. To date, there is obvious resistance to ACP in Poncirus and among some distant relatives of Citrus. There is also at least a little ACP resistance within most of the Citrus groups but, in general, there is no evidence that any Citrus germplasm would offer as much genetic resistance to ACP as Poncirus. Among trifoliate hybrids, there is some material that appears as resistant as Poncirus itself, indicating that traits of interest (greatly reduced oviposition, reduced longevity) were genetically inherited.
Construct optimization: We have engineered several new constructs for transformation in citrus genotypes. These include a ProBs314TBB-avrBs3: avrGf2 and a ProBs34TBB-avrBs3: avrGf2. In transient assays, these constructs elicited an earlier and stronger HR than constructs carrying avrGf1. We have also designed a construct which utilizes a portion of the promoter region of a citrus gene that demonstrates strong binding activity by PthA4 homologues. Transient assays demonstrate higher levels of activation than other constructs. Six new constructs are being used for stable transformation The constructs vary based upon the promoters used, the number of copies of the avrGf2 gene (single or multiple), the presence or absence of a terminator – nopaline synthase terminator (NOS T) upstream of the promoter and the plasmid used. Transformation summary In vitro germination experiments are ongoing with citrus varieties ‘Duncan’ grapefruit, ‘Ruby Red’ grapefruit and ‘Pineapple’ sweet orange for future epicotyl experiments. To date both epicotyl and cotyledon transformation experiments have been carried out with ‘Duncan’ grapefruit and ‘Pineapple’ sweet orange segments and the 6 new constructs designed with the avrGf2 gene. Sweet orange epicotyl and cotyledon transformation experiments have been carried out and a total of 1, 005 and 554 segments, respectively transformed with 5 of the 6 constructs. On the other hand 6 constructs have been used to transform altogether 3,623 grapefruit epicotyl segments and 5 constructs to transform 527 cotyledon segments in the transformation experiments. Shoots regenerated from transformed segments of sweet orange (53) and grapefruit (19) have been placed on rooting media. Putative sweet orange and grapefruit transgenic plants, 44 and 19 respectively originated from the regenerated shoots placed on rooting media have been placed in soil for acclimatization and will be tested via PCR for confirmation of integration of the transgene.
In our last progress report (9/15/2012), we reported the identification two citrus genes, temporarily named CtHRT1 and CtHRT2, capable of inducing hypersensitive response (HR)-like cell death when overexpressed in Nicotiana benthamiana. We also found that CtHRT1 and CtHRT2 belongs to a highly conserved family, in which other members from rice and Arabidopsis all can induce similar HR-like cell death in Nicotiana benthamiana. CtHRT1 and CtHRT2 are now renamed XBCT31 and XBCT32. Over the past three months, we carried out experiments to confirm the observed cell death response. An Arabidopsis protein with similar structure, but lower sequence identity, to XBCT31 and XBCT32 was expressed in Nicotiana benthamiana. No cell death was observed, indicating that the citrus XBCT31 and XBCT32-triggered tissue collapses are specific. HR cell death is often associated with electrolyte leakage caused by membrane damage. Ion leakage assays were performed to quantify the cell death. We found that significant amounts of ion leakage were detected 48 and 72 hours after infiltration with the XBCT31 or XBCT32 harboring Agrobacterium. In the infiltrated leaf discs, the development of visible tissue collapse kinetically correlated with the time course of ion leakage. Ion leakage was not induced by agroinfiltration of the empty vector. To confirm XBCT31 and XBCT32 accumulation, we carried out protein blot analyses. Leaf samples were harvested 40 hours after infiltration, at which time the necrotic phenotype was not visible. The XBCT31 and XBCT32 proteins were readily detectable in the infiltrated leaves. Interestingly, XBCT31 accumulated to a significantly lower level as compared with XBCT32. In contrast, a much more severe tissue death was induced by XBCT31. Therefore, XBCT31 appears to be a stronger cell death inducer, whereas XBCT32 seemed to have a weaker capability. Despite this difference, our data confirm the cell death activities of XBCT31 and XBCT32, and may be indicative of an activation of defense mechanisms. A manuscript with the data reported above and previously has been submitted to an international journal. We are currently constructing plasmids harboring XBCT31 and XBCT32, respectively, for stable citrus transformation to eventually test resistance to citrus canker and greening.
USDA-ARS-USHRL, Fort Pierce Florida has thus-far produced over 2,750 scion or rootstock plants transformed to express peptides that might mitigate HLB, and many additional plants are being produced. The more rapidly this germplasm can be evaluated, the sooner we will be able to identify transgenic strategies for controlling HLB. The purpose of this project is to support a high-throughput facility to evaluate transgenic citrus for HLB-resistance. Non-transgenic citrus can also be subjected to the screening program. CRDF funds are being used for the inoculation steps of the program. Briefly, individual plants are caged with infected psyllids for one week, and then housed for six months in a greenhouse with an open infestation of infected psyllids. Plants are then moved into a psyllid-free greenhouse and evaluated for growth, HLB-symptoms and Las titer. This report marks the end of the second quarter of the project, during which we have established the infrastructure for the screening program. A technician dedicated to the project has been hired, two small greenhouses for rearing psyllids have been completed and are functioning well, and 18 individually caged CLas-infected plants are being used to rear ACP for infestations. Psyllids will be available for challenging test plants in January. This screening program supports two USHRL projects funded by CRDF for transforming citrus.
We have transformed the cloned CtNPR1 (also named CtNH1) into the susceptible citrus cultivar ‘Duncan’ grapefruit. After survey on transgene expression, we now focus on the three lines, CtNH1-1, CtNH1-3, and CtNH1-5, which showed normal growth phenotypes, but high levels of CtNH1 transcripts. The three lines were inoculated with Xac306. They all developed significantly less severe canker symptoms as compared with the ‘Duncan’ grapefruit plants. To confirm resistance, we carried out growth curve analysis. Consistent with the lesion development data, as early as 7 days after inoculation (DAI), there is a differential Xac population in the infiltrated leaves between CtNH1-1 and ‘Duncan’ grapefruit. At 19 DAI, the level of Xac in CtNH1-1 plants is 104 fold lower than that in ‘Duncan’ grapefruit. These results indicate that overexpression of CtNH1 results in a high level of resistance to citrus canker. Overexpression of AtNPR1 or its orthologs is often associated with a constitutive expression of some PR genes. We therefore carried out RNA blot analyses to detect levels of chitinase 1 (Chi1). High levels of Chi1 were found in all three CtNH1 overexpression lines, which is in contrast with an undetectable basal level of this gene in wild-type plants. These results indicate that Chi1 is constitutively expressed in the CtNH1 overexpression plants. Because induction of Chi1 has been correlated with a resistance response in citrus (Porat et al. J. Plant Physiol. 158: 1585), our observations suggest that overexpression of CtNH1 leads to a constitutive activation of defense in citrus.
A talented postdoctoral fellow has been hired for the project and he has quickly begun making progress on research goals. Objective 1: Generate functional EFR variants (EFR+) recognizing both elf18-Xac and elf18-CLas. A. Mutagenesis of Arabidopsis EFR Conditions for optimal PCR random mutagenesis have been established and performed on the ectodomain of EFR. A library of approximately 1×106 clones, ready for transformation and screening, has been produced. A screening protocol has been established, whereby pools of 10 A. tumefaciens clones and infiltrated into N. benthamiana, and then scored for ROS induction in response to elf18-CLas. Screening has been initiated and we intend to screen approximately 3000 clones per week over the next three months. B. Natural variants of EFR Initial screening of a five species and cultivars of Brassicaceae has been performed, and we intend to obtain and screen a larger collection of species from several different genera for additional screening. Objective 2: Generate functional XA21-EFR chimera (XA21-EFRchim) recognizing axYS22-Xac. The PAMP receptors XA21 (from monocots) and EFR (from dicots) have been used to construct chimeric PAMP receptors. EFR-XA21 constructs have been produced to test the effectiveness of the XA21 cytoplasmic domain in signaling in dicots. This construct produces a ROS burst in response to elf18, to a similar degree as wild type EFR, when expressed transiently in N. benthamiana. Conversly, XA21-EFR and XA21 constructs have been produced and tested for responsiveness to ax21 in N. benthamiana. Thus far, a significant response has not been observed in the ROS burst assay; most likely due to a known issue of poor activity of the synthetic peptide (personal communication from Pam Ronald’s lab). Yet importantly, extracts from Xcv produced significant ROS burst with both XA21 and XA21-EFR constructs. Further evaluation of ax21 responsiveness using extracts from Xanthomonas euvesicatoria 85-10 (formerly, Xanthomonas campestris pv. vesicatoria) wild-type, or ax21 and raxST knockout strains (provided by Pam Ronald’s lab) that will enable us to conclude definitively on the functionality of XA21 and XA21-EFR is in progress. Transgenic Arabidopsis plants are being produced with XA21 or XA21-EFR to assess resistance to Xanthomonas campestris pv. campestris 8004 in dicots.
The overall goal of this project was to transfer disease resistance technology from Arabidopsis to citrus. Two specific aims were proposed in the original proposal. One was to overexpress the Arabidopsis MAP kinase kinase 7 (MKK7) gene in citrus to increase disease resistance (Transgenic approach), and the other was to select for citrus mutants with increased disease resistance (Non-transgenic approach). For specific aim #1, we have generated not only transgenic citrus plants overexpressing MKK7 but also transgenic plants overexpressing several other Arabidopsis disease resistance genes including NPR1, NAC1, MOD1, and EDS5. While disease resistance test for most of the transgenic plants is underway, transgenic plants overexpressing NPR1 were found to have increased resistance to citrus canker (see below). For specific aim #2, we have tested different citrus plant materials for mutagenesis, including calli, hypocotyls, and seeds. Chemical genetic screens have been carried out using these materials. In the last year of the project, we started a direct genetic screen for citrus greening-resistant varieties using grapefruit seeds mutated with gamma ray irradiation. This screen is still ongoing. During the project, we not only tried to accomplish the originally proposed work, but also explored the recently discovered disease resistance technology in the model plant Arabidopsis. At the end of the project, several significant results have been obtained. (1) We found that overexpression of the Arabidopsis NPR1 gene, which is a key regulator of systemic acquired resistance (SAR), in citrus increases resistance to citrus canker. This result has been published in European Journal of Plant Pathology. Furthermore, we found that the transgenic plants overexpressing NPR1 did not have increased resistance to citrus greening. (2) We found that the citrus canker-causing bacterial pathogen Xanthomonas citri subsp. citri (Xcc) is a nonhost pathogen of the model plant Arabidopsis. We discovered that Xcc neither grows nor declines in Arabidopsis, but induces strong defense gene expression. This result has been published in PLoS ONE. (3) Using the Arabidopsis-Xcc pathosystem, we found that the salicylic acid (SA) signaling pathway contributes to nonhost resistance against Xcc in Arabidopsis. Several genes of the SA signaling pathway were found to contribute to nonhost resistance against Xcc. (4) We found a group of novel genes, which play critical roles in nonhost resistance against Xcc in Arabidopsis. We revealed that Xcc grows significantly more in mutants of these genes. For instance, in one of these mutants, Xcc grows about 50-fold more than in the wild type, suggesting that the corresponding gene is a critical regulator of nonhost resistance against Xcc. More importantly, we found that overexpression of this gene confers resistance to several virulent bacterial pathogens; therefore, the newly discovered nonhost resistance genes hold great potential for generating disease-resistant citrus varieties. (5) We found that exogenous NAD+, which induces strong SAR in Arabidopsis, activates strong resistance to citrus canker, suggesting that the NAD+-mediated defense signaling pathway is highly effective against citrus diseases. Therefore, components we have identified in the NAD+-mediated signaling pathway could be used to engineer resistance to citrus greening and/or canker.
This is a 4-year project with 2 main objectives: (1) Over-express the Arabidopsis MAP kinase kinase 7 (AtMKK7) gene in citrus to increase disease resistance (Transgenic approach). (2) Select for citrus mutants with increased disease resistance (Non-transgenic approach). For objective 1, the transgenic citrus plants overexpressing the Arabidopsis MKK7 (AtMKK7) gene are under disease resistance test for citrus canker and greening. While waiting for the resistance test result, we expanded the project to identify genes that confer nonhost resistance to the citrus canker causing bacterial pathogen Xanthomonas citri subsp. citri (Xcc). We have previously established an Arabidopsis-Xcc pathosystem with the support of a USDA special grant, and have found that mutants of the SA signaling pathway are more susceptible to Xcc. These results have been published in PLoS ONE. Using the Arabidopsis-Xcc pathosystem, we screened available Arabidopsis mutants and identified a group of novel genes conferring nonhost resistance against citrus canker. Importantly, we found that overexpression of one of these nonhost resistance genes increases resistance to several virulent bacterial pathogens. Furthermore, we have generated citrus transgenic plants that express two salicylic acid (SA) biosynthesis genes. These transgenic plants are expected to accumulate more SA, which should transfer to stronger resistance to citrus canker and/or greening. We are testing the SA levels in the transgenic plants. For objective 2, we are continuing the direct genetic screen for citrus greening-resistant varieties. Gamma ray-irradiated Ray Ruby grapefruit seeds were germinated in soil and the resulting seedlings were inoculated with psyllids carrying greening bacteria. While adding more seedlings from gamma ray-irradiated Ray Ruby grapefruit seeds into the screen, seedlings developing greening symptoms were removed from the screen. We are watching the development of greening symptoms on the remaining seedlings.
This report covers the period of the last three months of 2012. Citrus Core Transformation Facility continued to operate at the high level and produced transgenic Citrus plants for multiple orders. The work continued toward a completion of ‘Y’ order and following transgenic Carrizo plants were produced: two plants with the gene from Y141 plasmid; 27 plants with the gene from the Y109 plasmid; and two plants with the gene from Y150 plasmid. Two Duncan plants were produced with the AZI1 gene. Two Duncan plants were produced with the gene from SF1 vector. Three Duncan plants were produced with the DPR1 gene. Continued experiments on some old orders yielded one Duncan plant with the EDS5 gene, four Duncan plants with the gene from WG20-7 vector, and five Duncan plants with the gene from WG19-5 vector. Eleven Duncan plants were produced transformed with genes from MOG800 vector. Seven Duncan plants were transformed with the AtBI gene. One Duncan plant with the CIV2 gene was also produced. The work on newer orders resulted in production of transgenic Duncan plants carrying genes from different vectors: four from the X4, ten from the X7, two from the X11, one from the X16, five from the X19, and five from the X20. The CCTF received six more new orders to produce transgenic plants carrying genes from vectors named pN4, pN5, pN7, pN9, pN12, and pN18. All of these orders requested production of transgenic Duncan plants. Cultures of Agrobacterium cells carrying these six binary vectors were already produced and are ready to be used in co-incubation experiments. With the plenty of recent and the newest orders, the facility will continue to operate at full capacity also working on full completion of older orders.
In this project, we proposed three aims in order to identify, characterize, and make use of citrus genes with a potential role in SA-mediated defense in engineering resistance to canker and greening diseases in citrus plants. Among the three proposed aims, the first aim has been completed, the second aim is about to be finished, and the third aim needs longer time to complete due to long-term growth nature of citrus plants. So far we have identified at least one citrus SA gene that could have effects on canker disease when overexpressed. Additional citrus transgenic plants are under further production and defense tests. We believe that we have met the expectations of the project and here provide a summary of the project. Objective 1: Identify genes positively regulating SA-mediated defense in citrus We identified over 10 citrus SA homologues via bioinformatics analysis. We used an RT-PCR approach to clone 10 full-length cDNA for the citrus SA homologues, which were further cloned into a binary vector pBIN19ARSplus for making transgenic plants in Arabidopsis and citrus. We have also finished collecting citrus tissues infected with Ca. L. asiaticus in a time course. qRT-PCR analysis with these samples was conducted for some SA genes. Our results showed that expression of at least one of the genes, ctNDR1, showed an induction upon HLB infection, suggesting a possible role of ctNDR1 in defense against HLB. Objectives 2: Complement Arabidopsis SA mutants with corresponding citrus homologues All 10 SA citrus genes were used to transform Arabidopsis plants, either complementing the corresponding mutants or overexpressing in wild type. We obtained T0 seeds for these constructs and selected most T0 seeds for T1 transgenic plants. Some seeds were further selected for homozygotes at the T2 generation. Most of the transgenic plants were tested for disease resistance to the infection of Pseudomonas syringae. So far, we found that at least two of the constructs ctNDR1 and ctEDS5 showed some level of disease resistance. However, there was no significantly increased resistance in CtNPR1 transformed Col or npr1-1 mutant and CtPAD4 transformed Col or pad4-1 mutant. Additional tests are undergoing for other transgenic plants. We have done more detailed characterization of ctNDR1 plants, which was summarized in a previous progress report (April 2012). A manuscript for this work should soon be submitted for a consideration of publication. Objectives 3: Assess the roles of SA regulators in controlling disease resistance in citrus We have so far produced transgenic plants for ctNPR1, ctEDS5, ctPAD4, and ctNDR1 and the presence of the transgenes in these plants were confirmed by PCR. In addition, we have tested disease resistance of ctNDR1 plants with Xanthomonas citri subsp (Xac), the causal agent for citrus canker disease, and found that overexpressing this gene confers some level of resistance to the strain. We will further test if ctNDR confers resistance to greening disease. In addition, we will continue to produce transgenic plants overexpressing other SA genes and selected transgenic plants will be tested for resistance to canker and greening diseases. These activities will be conducted after the end of the grant period.
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