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.
We now have citrus transformants in the greenhouse at UF for all of our constructs. PCR confirmations for all plants have been completed and indicate that from 17 to 88% of the surviving plants are positive for the R gene constructs. The least successful was the snc1 constitutive mutant expressed using the AtSUC2 promoter (2 positives out of 12 total). In addition, we have 11 (out of 15) citrus transformants containing the GusPlus reporter expressed using the AtPAD4 promoter, which we have recently found to be inducible by psyllid feeding. Our working hypothesis is that restricting expression of the R proteins to the phloem will lessen the negative impact that these proteins may have on normal growth and development. Transgenic citrus plants expressing either SNC1 or SSI4 wild type proteins using the phloem-specific AtSUC2 promoter appeared to exhibit a normal growth and leaf phenotype. This result was predicted since the respective R proteins should not have been activated in the absence of pathogens, and hence, would not trigger the hypersensitive response. In contrast, however, expression of the constitutively-active ssi4 protein in a phloem-specific manner resulted in approximately 60% of the plants with an obvious negative phenotype consisting of yellowish leaves, wavy-edged leaves, reduced internodal lengths, general stunting and leaf drop. This stunted phenotype resulted in death for 6 out of the 15 original transformants (40%). Although phloem-specific expression of the ssi4 mutant protein often (60% of the plants) resulted in either death or a negative growth phenotype, phloem expression of the snc1 mutant protein produced no abnormal phenotype. However, only 2 out of 12 plants were confirmed transgenics (AtSUC2/snc1). A similar tendency for the ssi4 mutant protein constructs to show an abnormal phenotype was also seen when expressed using the wound-inducible AtPAD4 promoter. The AtPAD4 promoter-snc1 construct showed no unusual phenotype. There are two questions that remain unresolved: 1) Why are some transformants showing a negative growth phenotype and others not, and 2) Do any of the R protein constructs confer resistance to Liberibacter? We are planning experiments that will evaluate the survival of Liberibacter in our transformed citrus lines. In addition, psyllid feeding tracks are being characterized histochemically to determine the relationship between AtPAD4/GusPlus reporter expression and the location of stylet sheaths and callose induction.
Analysis of transgenic citrus lines: We confirmed the presence of transgenes in transformed citrus plants using standard PCR techniques. In one line, SUC2-snc1 mutant (20-7), it was unusually difficult to establish that the desired construct was present. It required serial dilutions of genomic templates to reduce interference of the inhibitory substances present in plant genomic DNA extracts. This interference seemed to be correlated with the presence of this specific construct. It is possible that constitutively expressed snc1 mutant may affect the production of phenolic or other interfering compounds. In order to evaluate the survival of Liberibacter asiaticus (Las) in our transformed citrus lines, we first focused on the development of an assay to detect the presence of the bacteria in heavily infected, symptomatic citrus leaves. These were obtained from the UF Lake Alfred laboratory of Dr. William Dawson. Citrus leaves were sectioned into midveins and blades in order to determine the distribution of the infecting pathogen. Original quantities of tested materials were in the range of 40 mg. The primers used for PCR detection of the Liberibacter asiaticus were based on 16S ribosomal DNA, and as plant controls, the cytochrome oxidase COX gene was used (Pelz-Stelinski et al., 2010, J. Econ. Entomol. 103, 1531-1541). We were able to detect Las and Cox amplicons in genomic DNA isolates from these relatively small quantities of transgenic citrus leaf material: either in green (asymptomatic) or yellow symptomatic Las-infected leaves. Setting up a calibrated curve for real time quantitative PCR: Next, we generated Las and Cox PCR amplicons to be used in specific standard curves in the real-time PCR reactions to determine copy numbers of respective genes. The Wingless (Wg) gene that serves as the psyllid control was obtained from the genomic DNA isolated from 10 uninfected psyllids. Real-time PCR reactions required testing multiple variables in order to fine-tune the Las-detection assay, some being the primer and amplicon concentrations. We tested a range of amplicon concentrations from 10 ng to 1 pg (=12,190,283 copies), and in later experiments, down to 12 copies of Las, 14 copies of Cox and Wg. Also, lower concentrations of PCR primers were more optimal. As a starting point, we tested the expression of AtPAD4-GUSplus transgenic plants responsive to wounding to correlate the wounding event itself with the actual psyllid feeding. Preliminary citrus wounding experiments by slit-cutting, or needle-puncturing determined that the AtPAD4 promoter was very specifically induced by wounding. We performed numerous histochemical studies, including aniline blue, acid fuchsin, toluidine blue, Evans blue as individual and with combined staining, and fluorescence techniques to detect psyllid stylet sheaths. These were performed on cross-sections identified by GUS staining spots generated in response to psyllid-feeding (=wounding). After numerous attempts we were unable to establish this technology as a useful tool to meet our overall goal of the early Liberibacter detection in citrus plants.
A series of transgenics, produced in the last several years, continue to move forward in the testing pipeline. Currently, it appears prudent to replicate plants of each transgenic event and conduct challenges that last 10-14 months. Most of these plants in our program have been transformed with AMPs driven by several constitutive and vascular specific promoters. Several D4E1 lines appear to grow substantially better than controls even when infected, but do show the presence of CLas. Initial hopes of quickly identifying truly immune transgenics have matured to current efforts focusing on identifying significant resistance. For a disease that is devastating many Florida citrus groves, it is surprisingly difficult to get a consistent high-level of HLB disease even using known susceptible plants and inoculum sources with high levels of CLas. APHIS funded a grant, written by Gloria Moore, Ed Stover and Bob Shatters, focusing on identifying methods for highly-efficient and more standardized resistance screening, including exploration of strain x genotype effects. At the request of CRDF most FL researchers conducting research examining HLB-resistance met and shared observations and information. These data were summarized and used to finalize a set of experiments which are now underway. Jude Grosser and Ron Brlansky are playing key roles in addition to the grant authors. In our program, new constructs and resulting transgenics are in process, including hairpins to suppress PP-2 through RNAi (to test possible reduction in vascular blockage even when CLas is present), chimeral constructs that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab), and a citrus promoter driving citrus defensins (designed by Bill Belknap of USDA/ARS, Albany, CA).
A transgenic test site has been prepared at the USDA/ARS USHRL Picos Farm in Ft. Pierce, to support 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 more than twenty-one months. 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. USHRL has a permit approved from APHIS to conduct field trials of their transgenic plants at this site, with several hundred transgenic rootstocks in place: Dr. Kim Bowman has planted several hundred rootstock genotypes transformed with the antimicrobial peptide D4E1. An MTA is in place to permit planting of Texas A&M defensin transgenics produced by Erik Mirkov and his trees expressing the snow-drop Lectin (to suppress ACP) are now on the Stover permit. Information has been provided to complete the permit application by Eliezer Louzada of Texas A&M to plant his transgenics 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 will be 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. Additional plantings are welcome from the research community.
For this project we studied the response of ‘Carrizo’ citrange plants transformed with the Arabidopsis thaliana NPR1 gene (AtNPR1) to applications of salicylic acid (SA), L-flg22 peptide derived from Candicatus Liberibacter asiaticus (Originally this was not one of our objectives but we decided it was important to compliment our study) and HLB infection. In order to study and characterize this response we developed and standardized real time PCR assays for 21 citrus genes associated with the pathogen defense response: SA biosynthesis and signaling (AZI1, EDS5, ICS1, PAL1), PTI (PAMP-triggered immunity) and ETI (Effector-triggered immunity) (EDR1, EDS1, NDR1, PBS1, RAR1, SGT1), transcriptional regulation (NPR1, NPR3, R13032, R20540), the jasmonic acid pathway (COI1 and JAR1) and targets of the regulatory SAR pathway (BLI1, CHI1, PR1, PR1b, RdRp). A few genes (AZI1, CHI, EDS1, NDR1, NPR1, NPR2, PR1, SGT1, R13032 and RdRp) were generally differentially expressed between treated and control plants, serving as good indicators of plant defense in citrus. In total we studied 25 independent transgenic lines for their response to SA and L-flg22 to identify those with an enhanced response compared to wild type (non transgenic) plants. We also determined the expression levels of the transgene using Real time PCR. Certain lines had induction levels for some of the defense genes mentioned above several orders of magnitude higher than non transgenic lines (wild type controls). Infiltrations with L-flg22 was also very useful in discerning transgenic lines that could potentially be more tolerant to HLB due to their enhanced response. We further propagated using cuttings 20 of the most promising transgenic lines. This proved to be the most limiting part of our project as some of the lines took a long time to root and shoot (sometimes a year). Additionally some lines had high mortality rates. It would be interesting to figure out if this was due to the expression levels of the transgene. Due to limitations in space to conduct the HLB inoculation experiment we concentrated our efforts on 3 lines that showed the highest potential (much higher defense gene expression levels). We also included 2 lines with low or no expression of the AtNPR1 transgene and wild type controls. In total 26 plants are currently being studied for their response to HLB either as rootstocks of wild type scions or as wholly transgenics. This will show if transgenic AtNPR1 plants could potentially be used as rootstocks to control HLB in non-transgenic scions. We have started analyzing the plants for HLB infection. So far only those grafted on non-transgenic rootstocks have tested positive for HLB, however it is still too early to make any conclusions. Despite the fact that this project has ended we will continue monitoring the plants and will add more to the study as space and resources permit. However, several important outcomes have already been achieved with this project: 1) standardization of real time PCR assays a battery of defense genes that will allow the study of defense in citrus not only for HLB but for any other pathogen; 2) Proof that L-flg22 peptide can be used to study defense response in a shorter and more controlled way than graft inoculations; 3) AtNPR1 plants show an enhanced defense response compared to wild type plants.
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