Previously, we have shown that specific psyllid dsRNAs can be toxic to Asian citrus psyllids when the psyllids feed on citrus that have been engineered to produce these dsRNAs using a Citrus tristeza virus (CTV) expression vector. In this work, variability of dsRNA present within the tissues on which the psyllid is feeding effects toxicity to the psyllid and we have identified a threshold concentration of dsRNA needed to be toxic to adult psyllids. We have also identified a number of dsRNAs matching specific psyllid genes to which the psyllid are hypersensitive to (active at relatively low concentrations) when these dsRNAs are taken up orally through artificial diet feeding. Experiments have been initiated to express these dsRNAs in citrus and test their effects on all life stages of the psyllid when fed on these engineered plants.
The focus of this research has been to clone, express, and test the effect of suggested RNAi molecules on psyllid vectors using an artificial feeding system. We have developed a gut gene library, have isolated several sequences for critical proteins, and constructed RNAi molecules based on these sequences that will kill Asian citrus psyllids in controlled feeding experiments. We have also constructed and tested contest entrants. Several molecules have potential to control ASP. We have expressed two of the molecules in citrus using Dr. Dawson’s CTV vector, and have shown leaves from these trees are highly toxic to psyllids. There is a good correlation between RNAi expression and psyllid mortality. We will be testing other RNAi molecules using the CTV vector shortly. The sequences of the RNAi molecules and the genes they target are intellectual property. This project has given hope for a specific, environmentally friendly field control of Asian citrus psyllid without transforming trees. We are in the process of securing intellectual property rights for UF and USDA that will allow licensing, production, and marketing of this technology.
Cybrids are an asymmetric hybrid that contains the nucleus of one parent in combination with the mitochondrial and/or chloroplast genome of a second parent. Mitochondria and chloroplasts have a central regulatory role in integrating stress and/or programmed cell death signaling. Cybrids were evaluated according to the response to Xanthomonas citri spp. citri, (Xcc), The cybrids were created using the susceptible Red grapefruit (RedG, Citrus paradisi ) and the more tolerant Valencia orange (VO, Citrus sinensis) as a cytoplasm donor. The resistance inherited from VO is not a Hypersensitive Response (HR, qualitative) but instead a degree of quantitative resistance compared to highly susceptible Red Grapefruit. Evidence for this is based on an intermediate lesion phenotype for cybrids in vitro and in-planta. In contrast to development of callus in susceptible RedG, the inoculated area develops more necrosis by 15 days post inoculation. Xcc populations in the cybrids plateau at a level below populations in RedG, and the number of Xcc bacteria recovered from leaf disk was 10 fold lower than in the higly susceptible parent RedG. The lesion type is a mixture of necrotic and callus tissue that indicates that some cell death occurs and arrests the proliferation of Xcc. Expression of HR- and host pathogen interaction related genes in the RedG cybrids was is intermediate between VO and RedG. The different pattern of gene expressions suggests an interaction between the parent nuclear genes with the heterologous mitochondria and chloroplast from the cytoplasm donor (VO). The response to biotic stress caused by Xcc in citrus cybrids may be inherited at different levels depending which sets of genes contained in the mitochondrion or chloroplast genomes are transferred to RedG in the cybridization process.
New hybrids were created and entered into testing for development of promising new rootstocks and scions. Fruit quality, yield, tree size, and health data were collected from numerous rootstock and scion trials each year and summaries presented to grower groups on many occasions, including Indian River Citrus Seminar and Florida Citrus Show. Detailed fruit quality data collection continued from a large grapefruit rootstock trial in Indian River County at multiple harvest times to assess the influence of Sour orange, Swingle, US-812, US-942, US-897, US-852, and X-639 on grapefruit quality early, middle, and late in the season. Significant differences were observed among the rootstocks. Trees were propagated and placed into several new rootstock and scion field trials. Several established field trials were chosen for focused study of rootstock effect on tree tolerance to HLB, and trees were periodically tested for HLB and carefully monitored for growth, symptom development, and cropping. Differences in tree health as influenced by rootstock were noted, but those differences were relatively small. Cooperative work was begun 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 and high pH soils, using carefully controlled tests in the greenhouse. Three separate field studies were conducted to test SuperSour and other new rootstock selections for tolerance to Phytophthora-Diaprepes Complex. Field trials were planted to assess graft compatibility of SuperSour selections. Studies continue to assess citrus germplasm tolerance to Liberibacter – Huanglongbing (HLB) in the greenhouse and under field conditions. Collaborative work was conducted to study gene expression and metabolic changes associated with susceptible and tolerant plant responses to HLB, and to define genetic characteristics needed to prevent infection or avoid the damaging effects of the disease. Research was conducted to compare HLB tolerance of commercial rootstocks, with special focus on several hybrids of trifoliate orange. Preliminary testing was begun to evaluate the effect of grafting height on tree tolerance to HLB, when the rootstock is a tolerant variety. Other work continued to study the HLB tolerance of trifoliate orange hybrids and ways in which it might be used to create tolerant field trees. A study describing the tolerance of US-897 to HLB was published in the journal ‘HortScience’. A detailed study of gene expression changes in susceptible and tolerant citrus in response to HLB infection was published in the journal ‘Plant Science’. A study that demonstrated HLB was not transmitted through seed was published in the journal ‘HortScience’. A study that compared HLB tolerance of different rootstocks for sweet orange was published in the journal ‘Scientia Horticulturae’. Research accomplishments were documented in other scientific publications and presentations of research at ASHS Conferences, FSHS meetings, Plant and Animal Genome Conferences, Citrus Health Research Forum and other scientific meetings. Promising new scion cultivars were released, including seedless Pineapple and the seedless mandarin cultivar ‘Early Pride’. Cooperative trials continued and new trials established to provide more information on new scion performance and pollination effects. The new hybrid rootstock US-942 was released for commercial use because of outstanding performance in many trials. Seed of US-942, US-897, US-812, and US-802 was provided to the Florida Citrus Nursery Association for managed distribution to commercial nurseries.
Over the past year, our research has focused on the following areas: (i) Isolation and sequencing of TAL effectors from additional citrus canker strains Sequencing of TALE genes is especially difficult due to the presence of between14 and 20 repeats of the highly sequence-related DNA binding domain. However with considerable effort, we have now determined the sequences of eight proteins from five novel strains: A44 (Argentina), Etrog (Florida), 2090 (Florida), Miami (Florida), and 93 (Brazil), with four more protein sequences nearing completion. Although these strains show variation in phenotype or host range, our engineered promoter constructs containing 14 TALE recognition sites conferred recognition to TALEs in four of the strains, with the fifth pending analysis. These results further support our aim of engineering a resistance construct that will be triggered by a broad range of canker strains. Differences do occur in some of the sequences, and we plan to investigate how these differences may influence the behavior of strains in various assays. (ii) Production and testing of stable transgenic citrus lines: As of the end of this year, we have transformed a total of 21,504, 747, and 173 explants of ‘Duncan’ grapefruit, ‘Ruby Red’ grapefruit, and sweet orange cultivars, respectively, and have 446 plants in 4 inch pots. We have tested epicotyl and cotyledon explant material and find that epicotyls are the most efficient material for use with grapefruit, whereas cotyledons appear to work best for sweet orange. The Ruby Red cultivar is the most difficult to work with, because of the difficulty in sourcing seed. Each transformant is grown through shooting, rooting and transfer to soil, and then it is analyzed by PCR for each of the construct components – promoter, gene and selectable marker. Plants are further tested by pathogen inoculation. All stable and transient transformations were made with eight distinct gene constructs and a negative control. Overall, we find the broadest and best induction using the 14 box promoter, relative to other promoter versions. We have tested three HR-inducing genes – the Bs3 gene from pepper, and the AvrGf1 and 2 genes from Xanthomonas We have not observed activity of Bs3 in citrus to date. AvrGf1 has worked well in transient assays, but we have not yet analyzed enough stable lines to identify reproducible disease resistance. We continue to test lines as they mature, and AvrGf2 lines will also be tested when they become available. (iii) A third gene option for conferring resistance The type 3 effector AvrGf2, identified from X. fuscans subsp aurantifolii strain C, is being tested as another resistance gene option because it has been observed to cause a more robust HR on grapefruit than AvrGf1. The coding sequence was fused with the Bs3- PIP14 box promoter and used in transient and stable transformation assays. In transient assays, the avrGf2 construct did confer a robust HR within 3 days as compared to 4 days using AvrGf1. Stable transformation experiments involving epicotyls of ‘Duncan’ grapefruit, ‘Ruby Red’ grapefruit, and sweet orange segments, had 42, 16 and 32 plants transferred to rooting media, respectively. More transformants are in the pipeline, and all will be subject to molecular characterization and testing.
In the third and final year of funding, Core Citrus Transformation Facility (CCTF) maintained its level of performance and produced transgenic citrus plants for many satisfied customers. Considering that the major goals of proposed project to increase the capacity of the transformation lab were met within the first year of funding, during the second and third year of project duration CCTF had a task to maintain the achieved level of operation. The number of experiments and the quantity of produced transgenic material were at the level projected in the grant proposal. By accomplishing these tasks, CCTF assisted other researchers in efforts to improve different citrus cultivars by increasing their resistance and/or tolerance to diseases. Newly placed orders for transgenic plants stayed at high level. Altogether, thirteen new orders were received for processing during the third year of funding: pY46-Carrizo; pY102-Carrizo; pY141-Carrizo; pY150-Carrizo; pCitIntra-Duncan; pAZI-Duncan; pAtBI-Duncan; pBCR2-Duncan; pDPR1-Duncan; pLP1-Hamlin; pLP1-C-mac; pLP2-Hamlin; pLP2-C-mac. During the last quarter of this funding year, work was mostly concentrated on recent orders. Fourteen Duncan plants were produced carrying a gene of interest from the p35S-TRX vector and 23 more Duncan plants were produced carrying a gene from the pSucTRX vector. Multiple Duncan plants were produced toward satisfaction of ‘WG’ group of orders: eight-pWG22-1 plants, three-pWG21-1, and four pWG25-13 plant. Also, following Carrizo plants were produced for the ‘Yale’ order: nine plants with the gene from the pY46 vector and 11 plants with the gene from the pY102 vector. Eighteen Duncan plants were produced after treatment with bacteria harboring pBCR2 vector. Three more Duncan plants were produced with the EDS5 gene and six Mexican limes with the P35 gene. Four additional Duncan plants carrying a gene from pSUC-CitNPR1 were produced. In the previous three quarters of this year, small number of plants was produced for completion of older orders including: pNAC1 (1 plant); pMKK7 (1 plant); p33 gene (3 plants); pSUC-CitNPR1 (10 plants); p7+ p10 gene (32 plants). Most of the work was done on orders received in the last quarter of the second year and those placed in the third year of funding. The latter include: 12 Duncan plants (pWG19-5 vector); 11 Duncan plants (pWG20-7 vector); 18 Duncan plants (pWG21-1 vector); 22 Duncan plants (pWG22-1 vector); seven Duncan plants (pWG24-13 vector); 17 Duncan plants (pWG25-13 vector); 15 Duncan plants (pWG27-3 vector); 36 Duncan plants (ELP3 gene); 23 Duncan plants (ELP4 gene); nine Duncan plants (EDS5 gene); 10 Hamlin plants (pLC220 vector); 26 Duncan plants (p35 gene); 11 Duncan plants (35S-TRX vector), two Duncan plants (SUC-TRX vector). Joint efforts of Citrus industry and academic institutions to find solutions against huanglongbing (HLB), canker, and other citrus diseases are getting stronger with some positive results already being published. Continued funding for CCTF which is an integral part of this community and contributes greatly towards common goal will allow for the progress to go on by keeping production of transgenic material un-interrupted and at high levels.
In the 3rd and final year of the project, significant progress was made on several fronts. Juvenile Explant Transformation Protocol R&D: Key components of our transformation system were investigated in order to improve transformation and regeneration efficiency. The best medium for citrus transformation was determined to be the MS medium. Optimum hormonal levels in tissue culture medium was determined to be 3 mgL-1 BAP supplemented with 0.5 mgL-1 NAA for trifoliate rootstocks, Mexican lime and recalcitrant citrus cultivars like Volkamer lemon and mandarin / tangerines, and 1 mgL-1 BAP for sweet oranges and grapefruits. It was determined that a 3 hour soak in an auxin rich medium containing 1 mgL-1 2,4-D, and 0.5 mgL-1 NAA with 3 mgL-1 BAP significantly improved the transformation efficiency in a number of cultivars evaluated. Optical density of the bacteria was a determining factor in the genetic transformation of citrus. Trifoliate rootstock explants could tolerate a higher OD (0.6) while optimum transformation was observed at a lower OD for sweet oranges (0.15 or 0.3). We also observed co-cultivation duration was observed to be cultivar dependent. 3 days co-cultivation duration was observed to be optimum for cultivars with a thicker epicotyl such as trifoliate rootstocks or tetraploid selections. The optimum period for co-cultivation of sweet oranges was observed to be 2 days. Addition of 1mgL-1 GA3 resulted in rapid elongation of shoots, allowing in vitro micrografting within a month of initial selection of shoots. We determined that addition of Lipoic acid ‘ an antioxidant to shoot regeneration medium following transformation dramatically enhanced the transformation efficiency. This addition has resulted in improved transformation efficiency in otherwise recalcitrant cultivars. Transgenic plants from precocious sweet orange somaclones including OLL8, B4-79, Vernia 2-1, and a precocious mandarin W. Murcott, containing the LIMA antimicrobial construct, were produced and were grafted to precocious rootstocks (Amblycarpa+ Benton and Changsha + Benton somatic hybrids) for continued early flowering-induction experiments. Seasonal effects of on regeneration potential and transformation success rate were also evaluated, and confirmed that each cultivar behaved differently based on the time of fruit harvest and seed germination. A rapid ex vitro micrografting technique suitable for propagating in vitro and young ex vitro transgenic stem pieces was developed. Combining all of these advances is expected to cut in half the time from initiating an experiment to flowering and fruiting transgenic trees, thus making juvenile transformation more competitive with mature tissue transformation. In addition we have developed an efficient protocol using cell suspension cultures that has enabled us to transform seedless or recalcitrant cultivars such as the precocious mandarin cultivar W. Murcott and the seedless cultivar Okitsu Wase satsuma. This protocol has created an avenue for insertion of useful traits into any polyembryonic citrus cultivar that can be established as an embryogenic cell suspension culture. This research supported 8 journal publications. Transformation with Early Flowering Genes: We have regenerated many transgenic citrus plants that include: poplar FT gene behind either the 35S or heat shock promoter; co-transformed Carrizo plants with two cassettes, one containing 35S-cft1 and the other containing AtSUC2 ‘ gus; and numerous transgenic plantlets of Hamlin and Carrizo containing P27, P28, P29, PATFT and pPTFT. All of these plants are at various stages of evaluation.
Objective1 (Characterization of the resistance to citrus canker): A comparative study of grapefruit (C. paradisi) cv. Duncan, a very susceptible host, and two resistant cultivars of kumquat (Fortunella spp.), ‘Meiwa’ and ‘Nagami’, was conducted to evaluate the mechanisms involved in resistance of kumquat to the citrus canker. To expedite and standardize the evaluation of resistance in citrus genotypes, a prototype needle-free device was designed to inoculate detached leaves and attached leaves. Xcc inoculum densities of 105 and 108 cfu/ml were infiltrated into immature leaves in vitro and in the greenhouse. At higher bacterial inoculum density, kumquat cultivars developed a hypersensitive (HR)-like reaction in the infiltrated area within 3-4 da. At the lower inoculum density no symptoms or a few small necrotic spots were formed in at 15 days post inoculation (dpi). Susceptible grapefruit infiltrated with the same inoculum densities produced no visible tissue alterations at 3 dpi and required 7 to 15 days to develop water-soaking, hypertrophy and hyperplasia typical of canker lesions in compatible hosts. Phenotype of the lesions, bacterial population growth, anatomical changes in the infiltrated tissue and early expression of genes related to programmed cell death in kumquat were indicative of HR that reduced growth of Xcc in the inoculation site and restricted development of infection. Objective 2 (Characterization of citrus cybrids and comparison with parental genotypes): Highly susceptible Red grapefruit (RG) produced abundant lesions at 15 dpi with cellular hypertrophy and hyperplasia typical of callused lesions in compatible hosts. Similar lesion phenotype was observed in detached leaves and attached leaves. Valencia orange (VO) had fewer callus-like lesions, and more necrotic lesions. Numbers of lesions was greatest for RG (93) and least for VO (47). The cybrids of RG+VO showed a variable number and phenotype of the lesions, 4 cybrids developed a higher number of lesions than VO (>50), 11 cybrids produced an intermediate number (25-50), and 5 cybrids formed a lower number of lesions than VO (<25). In contrast to the callus-like lesions in RG, lesions in the less susceptible cybrids were more necrotic as observed for VO. Thus, canker resistance appeared to be quantitatively inherited from VO based on the ivariation in lesion phenotype among the cybrids. This was confirmed by Xcc population growth in Cy 3 and Cy 10 that was similar to VO and nearly one log unit lower than RG at 15 dpi. Objective 3 (Gene responses of cybrids to Xcc): Putative genes with mitochondrial and chloroplast-related function were identified from EST sequences in kumquats and grapefruit from the Objective 1 study. Responses of host-pathogen interaction genes associated with mitochondrial and chloroplast-related function were identified in VO and Cy 3 and Cy 10, which differed from RG. Pathogenicity related proteins PR4, chitinase (CHI) and beta-glucanase (BG) were up regulated at 4 and 24 hpi in both cybrids. Higher expression of heat shock proteins (Hsp20) in the cybrids suggested a differential interaction of genes from the nucleus with mitochondria and chloroplast genes from the cytoplasm donor. Expression of genes related to programmed cell death and development of hypersensitive reaction to plant pathogens, e.g., alternative oxidase (AOX), aconitase-iron regulated protein (IRP1) and ascorbate peroxidase (APX2) were up-regulated at 4 hpi in the cybrids, as evidence for enhanced antioxidant activity. The response of cybrids to Xcc may be expressed at different levels depending on whether mitochondrial and/or chloroplast genomes are transferred in the cybridization process. At the present time, the most promising cybrids are under propagation for field evaluation in areas with endemic citrus canker.
The antibody developer, Creative Biolabs, Inc., now has the purchase order from Penn State, which was required to initiate the project. The target peptide for NodT is now being synthesized. Screening of antibody libraries for high-quality antibodies is expected to take approximately 6 weeks, and was initiated on April 24, 2012. They plan to isolate about 5-20 antibodies with ability to bind the NodT protein.
At this stage, we have completed Aim 1 (Identify genes positively regulating SA-mediated defense in citrus) and most work described in Aim 2 (Complement Arabidopsis SA mutants with corresponding citrus homologs). We have so far cloned more than ten citrus SA genes, all of which are at various stages of gene transformation and analysis of transgenic plants. We are actively working on Aim 3 (Assess the roles of SA regulators in controlling disease resistance in citrus) to make citrus transgenic plants over-expressing the SA genes and to assay the plants for resistance to HLB and citrus canker diseases. So far we have confirmed transgenic citrus expressing ctNDR1, ctPAD4, and ctEDS5. Additional constructs are in the pipeline of citrus transformation. While the cloned citrus SA genes are at various stages of analysis, the most advancement that has been made so far is with ctNDR1. We have obtained data to support that manipulating the level of ctNDR1 could lead to enhanced disease resistance. The main results on ctNDR1 are summarized below. We are in the middle of preparing a manuscript for publication. 1. Overexpression of CsNDR1 could complement the Arabidopsis mutant ndr1-1 for its disease susceptibility to and the lack of hypersensitive response to Pseudomonas syrinage avrRpt2 infection. ctNDR1 conferred resistance is largely dependent on the expression of ctNDR1 (dosage dependency) in Arabidopsis 2. The Arabidopsis NDR1 was previously shown to act downstream of a subset of resistance genes (i.e. RPS2 that recognizes avrRpt2) but not required by other resistance genes, such as RPS4. However, we found that ctNDR1 overexpression increases resistance to both P. syringae strains expressing avrRpt2 or avrRps4, suggesting the activation of general defense mechanism in the ctNDR1 overexpression plants. 3. Consistent with enhanced disease resistance to different pathogens, we found that higher expression of ctNDR1 also led to increased accumulation of salicylic acid, a key signaling molecule that activates broad disease resistance. 4. We performed quantitative RT-PCR analysis of NDR1 in mock-inoculated and Ca. L. asiaticus-inoculated ‘Cleopatra’ mandarin seedlings. We found that expression of ctNDR1 was inducible by HLB in two independent experiments (experiment 1 with 32 week-old seedlings and experiment 2 with 30 week-old seedlings). These results suggest a potential role of ctNDR1 in HLB resistance. 5. We have so far obtained 29 independently transformed transgenic citrus plants carrying ctNDR1 overexpressing construct. The presence of the transgene was confirmed by transgene-specific primers. We should begin to test these plants for resistance to citrus canker disease in the next few months.
Our progress for the current quarter is as follows: 1. Transgenic Duncan grapefruit lines expressing avrGf1 driven by the Bs3- PIP14 box promoter are currently being tested for resistance by spray inoculating X. citri subsp citri 306 at 10^8 cfu per ml suspended in sterile tap water on young citrus leaves. Resistance confirmed by transgenic lines will be assessed by evaluating canker lesions between transgenic and non-transgenic control plants. 2. Compared to AvrGf1, the type 3 effector AvrGf2, identified from X. fuscans subsp aurantifolii strain C, seems to provoke a faster hypersensitive reaction on Duncan grapefruit. The avrGf2 coding sequence was fused with the Bs3- PIP14 box promoter into the pK7FWG2 binary vector and transformed into A. tumefaciens strain Agl-1. In planta transient expression in Duncan grapefruit was assessed for induction by X. citri subsp citri 306 TALEs using the pathogen inducible promoter (Bs3- PIP14 box):avrGf2 construct for citrus canker resistance. Upon activation of the construct by Xcc TALEs, the expressed AvrGf2 elicited a resistance response that was faster and more effective than that of a Bs3-PIP14 promoter construct driving AvrGf1. 3. Transgenic Duncan grapefruit carrying the AvrGf2 construct are in the development pipeline to produce stable transformants. Transfomation was performed on 2,049 grapefruit epicotyl segments. 4. Germination and epicotyl transformation experiments with Duncan grapefruit are continuing. Molecular characterization involving PCR is ongoing and used to confirm transgenic lines from the putative transgenic plants regenerated. To date, a total of 190 putative transgenic plants have been screened and 23%, 20% and 6% of the plants tested positive for the Bs3 promoter, nptII and the avrGf1 genes, respectively.
Results from four sweet orange rootstock field trials exposed to HLB were published. A detailed study comparing tolerance of rootstocks to HLB in the greenhouse was completed and results are being prepared for publication. The summation of these studies indicates there are significant differences in the tolerance of different rootstocks to HLB, and that, under some conditions, this may have a significant effect on tree growth, health, and performance. Depending on the conditions, rootstocks that sometimes showed increased tolerance to HLB included Volkamer, US-897, US-802, US-942, US-812, and Carrizo. Fruit quality, yield, and tree size data were collected from five late-season rootstock field trials. Detailed fruit quality data collection continued from a large grapefruit rootstock trial in Indian River County at multiple harvest times to assess the influence of Sour orange, Swingle, US-812, US-942, US-897, US-852, and X-639 on grapefruit quality early, middle, and late in the season. Significant differences were observed among the rootstocks. Cuttings were made of 100 supersour selections in preparation for testing with Diaprepes/Phytophthora and HLB, and also for field trials with commercial scions. A supersour rootstock trial with Hamlin scion and 500 trees 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 and high pH soils, using carefully controlled tests in the greenhouse. Studies continue to assess citrus germplasm tolerance to Liberibacter – Huanglongbing (HLB) in the greenhouse and under field conditions. Studies to identify the metabolic changes associated with HLB disease development have been completed and a manuscript is being prepared for publication. Recognizing the clear tolerance of some citrus germplasm to HLB, the second half of a study is underway to define the interaction of rootstock tolerance/susceptibility with scion tolerance/susceptibility. Another study has begun to assess the additional benefit of expanding the amount of a tree that is the HLB-tolerant rootstock to include the trunk and scaffold branches. Collaborative work continues to study gene expression and metabolic changes associated with susceptible and tolerant plant responses to HLB, and to define genetic characteristics needed to prevent infection or avoid the damaging effects of the disease. Greenhouse and field studies are continuing to determine the most efficient methods to evaluate new citrus germplasm from crosses and transformation for resistance or tolerance to HLB. Selected anti-microbial and citrus plant resistance genes were inserted into outstanding rootstock and scion cultivars to develop new cultivars with increased resistance to HLB. Research is continuing to use HLB responsive citrus genes and promoters identified in the gene expression study published last year for inducing or engineering resistance in citrus. Fifteen new transgenic rootstocks with selected antimicrobial genes were propagated and entered into controlled greenhouse tests to assess tolerance to HLB. A field trial continued with selected transgenic rootstocks. Collaborative work continued to assess rootstock interaction with scion, nutrition, and management factors in determining tree tolerance to HLB.
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 the transgenic approach proposed in objective 1, besides transforming the Arabidopsis MKK7 (AtMKK7) gene into citrus, we are making transgenic citrus plants overexpressing SA biosynthesis genes. We expect that citrus transgenic plants overproducing SA would have increased resistance to citrus canker. Although exogenous application of SA does not increase resistance to citrus greening, increasing endogenous SA levels may have different effect. Transgenic citrus plants expressing the Arabidopsis MKK7 (AtMKK7) gene are currently under canker resistance test. We have propagated these plants for citrus greening test. We are trying to generate citrus transgenic plants overexpressing several other Arabidopsis disease resistance genes including ELP3 and ELP4. The mutant screen proposed in objective 2 has been continued. More gamma ray-irradiated Ray Ruby grapefruit seeds have been gamma ray irradiated. Part of the seeds will be plated into large glass Petri dishes as well as Magenta boxes containing water agar. Shoots formed on the seeds previously plated will be transferred onto selective medium containing 0.2 mM of sodium iodoacetate. Some shoots formed on these gamma irradiated seeds have been screened again on the selective medium. Part of the seeds will be directly sown into soil, and seedlings from these seeds will be test for greening resistance. We would like to test whether a direct genetic screen could work for identifying citrus greening-resistant varieties. We will continue to germinate gamma ray-irradiated Ray Ruby grapefruit seeds in soil and inoculate the seedlings with psyllids carrying greening bacteria. We have been watching the development of greening symptoms on the seedlings.
The objectives of this project include: (1) Characterization of the transgenic citrus plants for resistance to canker and greening; (2) Examination of changes in host gene expression in the NPR1 overexpression lines in response to canker or greening inoculations; (3) Examination of changes of hormones in the NPR1 overexpression lines in response to canker or greening inoculations; (4) Overexpression of AtNPR1 and CtNPR1 in citrus by using a phloem-specific promoter. We searched the citrus genome database (http://www.phytozome.org/citrus.php) with BLAST and identified nine genes similar to AtNPR1 or its Arabidopsis homologs. Among them, CtNPR1 (also named CtNH1) is the most closely-related to AtNPR1 based on phylogenetic analysis, supporting an orthologous relationship. The Figwort mosaic virus (FMV) promoter was used to overexpress CtNH1 in citrus. Previous studies in soybean showed that the FMV promoter is significantly stronger than the Cauliflower mosaic virus (CaMV) 35S promoter for gene expression (MPMI 21: 1027). Three lines, CtNH1-1, CtNH1-3, and CtNH1-5, which showed normal growth phenotypes, but high levels of CtNH1 transcripts have been identified. When inoculated with X. citri subsp. citri (Xcc), 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 Xcc 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. CtNH1 plants have been propagated by grafting and are inoculating with Candidatus Liberibacter asiaticus (Las) in two laboratories. A microarray experiment was conducted using CtNH1 and non-transgenic Duncan grapefruit inoculated with Xcc. A needleless syringe was used to infiltrate the leaves with the bacterial culture (OD600 to 0.3). Three time points were used for this study. For each time point, three replications were used. Data analysis indicates that at p value <0.01, a total of 451, 725, and 2144 genes were differentially expressed at 6, 48, and 120 hours post inoculation (HPI), respectively. Using the visualization tool Mapman 3.5.1, the differentially regulated genes (Log FC ' 1 and Log FC ' -1) were mapped to give an overview of the pathways affected. Interestingly, at 120 HPI, a large number of genes involved in protein degradation and post-translational modification were differentially regulated. Furthermore, numerous genes involved in signaling also showed differential expression at this time. The results indicate that a large number of genes involved in the regulation of transcription were up-regulated in the transgenic plants at 120 HPI, and also at 48 HPI, although to a lesser extent. The photosynthetic pathway was affected to a larger extent at 48 HPI, which is signified by a large number of genes involved in photosynthesis being up-regulated in the transgenic plant when compared to the non-transgenic citrus. We have completed the SUC2::CtNH1 construct, in which CtNH1 is driven by a phloem-specific promoter from the Arabidopsis SUC2 gene. The construct were transformed into 'Duncan' grapefruit. To date, ten transgenic lines have been obtained. They are ready for Las inoculation.
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. We are now trying to identify the genotypic and phenotypic traits that promote resistance. Volatiles were collected using a SPME fiber and aerations from one genotype of P. trifoliata, one resistant and one susceptible genotype of xCitroncirus sp., and one susceptible control (Citrus macrophylla). The volatiles were analyzed using gas chromatography-mass spectrometry to identify whether the volatile profiles differ among susceptible and resistant plants. We found clear differences in the volatile profiles and are currently identifying all volatile compounds collected from the four samples. We will expand our sampling of volatiles by analyzing closely related genotypes of P. trifoliata and xCitroncirus sp. that differ in their susceptibility to ACP, which will allow us to identify the most likely compounds that attract or deter ACP. We are also planning a project that will allow us to analyze the amino acids, sugars, flavenoids, carrotenoids, isoprenoids, and sterols in phloem contents of closely related genotypes of P. trifoliata and xCitroncirus sp. that differ in their susceptibility to ACP. This will give us information about the underlying reasons why ACP avoid certain genotypes of citrus and help identify genes that can be used in citrus breeding programs to confer resistance to ACP. We also are screening grapefruit trees that have been genetically transformed to express Lectin from the snowdrop pea for susceptibility to ACP. We are comparing rate of oviposition, nymphal development, and lifespan of adult ACP on three varieties of grapefruit that express lectin and one variety that does not. Expression of Lectin does not deter oviposition by ACP. However, our first replication indicates that adult ACP likely have a shorter lifespan on the two varieties of grapefruit expressing the highest levels of Lectin. Additionally, nymphs die instead of reaching the adult stage on the variety expressing the highest level of lectin. A second replication of the experiments with adults and nymphal ACP is underway to verify these results. We also are starting an experiment to determine whether Lectin interferes with acquisition or transmission of citrus greening disease. ARS maintained contact with the Fujian Academy of Agricultural Sciences through emails and phone calls. During October 2011, ARS (Duan) visited FAAS in China and reviewed their research progress. FAAS initiated no-choice experiments with Poncirus accessions and added different accessions of P. trifoliata, B. koenigii and other species outside the genus Citrus to their field studies. To date ARS and FAAS findings are in general agreement about susceptibility/resistance of specific germplasm studied by each group, although some germplasm that is clearly resistant to ACP in Florida did not appear as susceptible in China (probably due to escapes). FAAS is evaluating some germplasm that ARS has not studied. Should any of this material appear to have ACP resistance, ARS will attempt to acquire it.
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