Based on good field performance and superior yield with severe HLB infection pressure on the east coast, the rootstocks US-1279, US-1281, US-1282, US-1283, and US-1284 were released by USDA for commercial use. Fruit yield of Hamlin trees on these rootstocks is 2-4 times the yield of trees on Swingle in the same trials, and the trees also have fruit that is larger in size and higher in sugar content. These promising new rootstock selections have been provided to Florida DPI for establishment of certified budwood sources, are being made available to the CRDF Product Development Project to establish large scale commercial field trials, and will also be used in multiple field trials with commercial growers with funding provided by the HLB-MAC project. A special permit has been obtained from Florida DPI to immediately begin establishing widespread commercial field trials using clean USDA sources of these and other new rootstocks. Based on this permit, cooperative arrangements are being made with commercial Florida nurseries for large scale vegetative propagation of these promising new rootstocks, as needed to meet commercial demand until adequate seed sources can be developed. A material transfer agreement was signed with Agromillora Catalana, SA, to immediately allow that company to begin rapid micropropagation of these most promising rootstock selections for use in Florida. Trees were prepared for planting of three new field trials with Supersour rootstocks later in 2014. About ten thousand new propagations of Supersour rootstocks were prepared for budding and planting in additional field trials in 2015. Work began to construct an additional greenhouse at USDA to propagate Supersour rootstocks for field trials. Cooperative work continued with a commercial nursery to multiply promising Supersour rootstocks to produce trees for medium-scale commercial trials. A Valencia field trial was planted with a commercial cooperator to evaluate performance of several promising Supersour rootstock selections alongside other good commercial rootstocks. Greenhouse studies continued to assess Supersour tolerance of CTV, calcareous soils, and salinity. Trees were planted into the field to establish seed sources for the most promising Supersour selections. A study of the interaction between rootstock tolerance and scion tolerance/susceptibility will be presented at the HLB conference in February and is being prepared for publication. This work provides considerable insight into disease progression and the potential for improved management. Studies continued on defense-related genes and small RNAs associated with HLB infection, in collaboration with University of Maryland and University of California research groups. A study of localized defense gene expression in shoots and roots provided evidence of striking differences that are a major advance in understanding and yield strong insights into ways to overcome the disease. A new field trial was planted to evaluate grafting height effect on tree tolerance to HLB. Two replicated tests with US-942 rootstocks that overexpress the citrus defense gene CtNDR1, are showing significant reduction in Las infection for some of the transformed clones. Monitoring and data collection continued on previous groups of transgenic plants that have been inoculated with HLB. Several transgenic rootstock selections showing increased resistance to HLB have been identified from groups transformed with other resistance genes, and are also being prepared for confirmation testing. One hundred new transgenic US-942 and Sour orange rootstocks were produced, targeting to increase tolerance to HLB by manipulation of the citrus resistance genes CtNHL3, CtNHO1, CtDIR1, CtAZL1, CtERF1, and CtFMO1.
USDA-ARS-USHRL, Fort Pierce Florida is producing thousands of scion or rootstock plants transformed to express peptides that might mitigate HLB. 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. This screening program supports two USHRL projects funded by CRDF for transforming citrus. 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 two weeks, 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. USDA-ARS is providing approximately $18,000 worth of PCR-testing annually to track CLas levels in psyllids and rearing plants. Additionally, steps to manage pest problems (spider mites, thrips and other unwanted insects) are costing an additional $1,400 annually for applications of M-Pede and Tetrasan and releases of beneficial insects. To date on this project, it funds a technician dedicated to the project, a career technician has been assigned part-time (~50%) to oversee all aspects of the project, two small air-conditioned greenhouses for rearing psyllids are in use, and 18 individual CLas-infected ACP colonies located in these houses are being used for caged infestations. Additionally, we established new colonies in a walk-in chamber at USHRL to supplement production of hot ACP. Some of the individual colonies are maintained on CLas-infected lemon plants while others are maintained on CLas-infected Citron plants. As of September 2, 2014, a total of 6,208 transgenic plants have passed through inoculation process. A total of 122,855 bacteriliferous psyllids have been used in no-choice inoculations.
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 four years. Dr. Jude Grosser of UF has provided ~600 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser planted an additional group of 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, and Ed Stover 50 sweet oranges (400 trees due to replication) 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. 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. So far, the 1/8th poncirus hybrid nicknamed Gnarlyglo is growing extraordinarily well. It is being used aggressively as a parent in conventional breeding. In a project led by Richard Lee, an array of seedlings from the Germplasm Repository are in place, with half preinoculated with Liberibacter. Additional plantings are welcome from the research community.
A chimeral construct that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab) is being tested. Many transformed Carrizo with the chimera AMP were obtained. Exposure to canker inoculum showed remarkabe resistance in chimera compared to control. Canker infiltration showed greatly increased resistance in two chimera AMP and several thionin transgenics, at 107CFU/ml. RNA was isolated from transgenic plants containing chimera and thionin. RT-PCR showed gene expression in the transgenic plants. Further gene expression level was evaluated with RT-qPCR. Our results showed gene expression variation between different transgenic lines, from several fold to 35 fold. Transgenic lines containing D4E1 were evaluated with Xcc infiltration. All the transgenic lines with canker development at 105 CFU/ml while some transgenic lines show less canker development at 104 CFU/ml. Bacterial growth rate in transgenic lines containing D4E1, chimera and thionin was investigated by qPCR. Our results showed some transgenic lines containing chimera and thionin had low Xcc growth rate. More transformed Hamlin carrying chimera were generated and over 30 were confirmed positive by PCR. About 20 Hamlin transformed with thionin also were obtained. They will be tested by RT-PCR and replicated for HLB challenge. Putative transgenic plants of PP-2 hairpins (for suppression of PP-2 through RNAi to test possible reduction in vascular blockage even when CLas is present) and of PP-2 directly are grafted in the greenhouse and growing for transgene verification, replication and testing. 40 putative transgenic plants transformed with citGRP1 were tested by PCR and twenty two of them were confirmed with citGRP1 insertion. RNA was isolated from some and RT-PCR showed gene expression. Some transgenics with over-expression of citGRP1 had increased resistance to canker by detached leaf assay and infiltration with Xanthomonas. Over 60 transgenic Carrizo with GRP2 were transferred to soil. DNA was isolated from 20 of them and 19 of them are PCR positive. Some of them showed canker resistance when infiltrated with Xcc at concentration of 105/CFU. Fifteen transgenic Carrizo and seven transgenic Hamlin with peach dormancy related gene MADS6 were planted in soil and they are ready for DNA isolation. To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was cloned into pBinARSplus vector Flagellins are frequently PAMPS (pathogenesis associated molecular patterns) in disease systems and CLas has a full flagellin gene despite having no flagella detected to date. The consensus FLS2 clone was obtained and used to transform Hamlin and Carrizo so that resistance transduction may be enhanced in citrus for HLB and other diseases. Many putative transformants were generated on the selective media. DNA was isolated from 80 of them: 38 Carrizo and 7 Hamlin are positive by PCR test. Reactive Oxygen Species (ROS) assay showed typical ROS reaction in three of transgenic Hamlin which suggest nbFLS is functional in citrus PAMP-triggered immunity. However, there is only slight canker resistance by infiltration test. Spray inoculation was tried and some of them show obvious canker resistance. To disrupt HLB development by manipulating Las pathogenesis, a luxI homolog potentially producing a ligand to bind LuxR in Las was cloned into binary vector and transformed citrus. Both transformed Carrizo and Hamlin were obtained. Further investigation are underway. A series of transgenics scions produced in the last several years continue to move forward in the testing pipeline. Several D35S::D4E1 sweet oranges show initial growth in the field which exceeds that of controls. A large number of ubiquitin::D4E1 and WDV::D4E1 plants and smaller numbers with other AMPs are replicated and in early stages of testing.
We have found that cell penetrating peptides (CPPs) can be used to deliver molecular ‘cargo’ (e.g. protein or DNA) into a variety of plant tissues, including those of citrus. However, to date, stable genetic transformation with CPPs but without the use of Agrobacterium-based DNA sequences has not been achieved in citrus. The Foundation wanted us to concentrate on this specific objective due to the considerable regulatory issues surrounding the use of such bacterial sequences in transgenic plants. Fortunately, since we began this project, there have been a number of new genetic developments. Targeted DNA modification, or ‘gene-editing’, methods such as Zinc Fingers and TALEN technology have been developed. However, the most recent and exciting developments have been with the CRISPR/Cas system. This system not only allows for very precise, targeted gene editing, but gene over-expression or repression is also possible. Further, the CRISPR/Cas system is simpler to engineer than other available systems, and less likely to have nonspecific binding or to recombine out. Finally, this methodology will satisfy the main objective, which should allow engineering of citrus without the use of inserted non-native (e.g. bacterial) DNA sequences. We hope that the CRISPR/Cas system will allow transgenic plants to be free from regulatory issues (although, the USDA still has this under consideration). This quarter, we have concentrated on designing CRISPR/Cas vectors for use with CPPs and citrus tissue. This is still underway.
This quarter we have continued to make progress on our transformation approaches: 1. Stable transformation in citrus using new vectors. We previously found that the original vector system used to create the Bs3 promoter constructs was contributing to low transformation efficiency in citrus, and switched to a pCAMBIA-based vector system. Two ProBs314EBE:avrGf2 transgenic plants created using the new vectors in citrus cultivar Carrizo have now been confirmed by PCR to contain the transgene, and these will be examined for appropriate gene expression by RT-PCR. An ongoing pipeline of transformants are being generated with the new vectors. To date a total of 2,056 putative transgenic shoots of grapefruit, sweet orange and Carrizo were screened for this period. Results show that no GUS positive has been observed for the sweet orange cultivar transformed with any of the constructs analyzed, however, 3 shoots were chimeric for the pCAMBIA2201:NosT:Bs3super::avrGF2 construct. In general, Grapefruit had a combined total of 12 and 41 shoots being GUS positive and chimeric for GUS, respectively for all constructs anlayzed while Carrizo citrange had 185 and 180 shoots being GUS positive and chimeric for GUS, respectively. These GUS and chimeric shoots will later be screened via PCR once rooted and transferred to soil for acclimatization. 2. Stable transformation in tomato test system: A tomato test system was previously designed and tested in which the 14 EBE promoter was fused to the avrBs4 gene capable of inducing a hypersensitive reaction in tomato. T1 generation of Bonny Best and Large Red Cherry transformed with ProBs3_14EBE:avrBs4 were screened for pathogenicity reaction with X. euvesicatoria strain (Race 9). Promising resistant transgenics have been selected for T2 generation analysis to confirm that this test system, in which resistance is induced by the effectors AvrBs3 and AvrHah1, is functional for conferring stably-transformed transgenic disease resistance.
We are still working to obtain stably transformed citrus containing the BS3 promoter with added TAL effector binding elements (4 or 14 EBE) fused to a defense response inducing gene. We have obtained three transformants with a 14 EBE construct driving either the AvrGf1 or AvrGf2 Xanthomonas effector gene in Carrizo citrange, a citrus variety more amenable to transformation. PCR-based analysis of gene expression demonstrated that these constructs were induced as expected upon infection with the virulent strain X. citri strain Xcc306, validating that the promoter works in stably-transformed citrus. Whereas expression of AvrGf1 or 2 genes in orange and grapefruit triggers a hypersensitive defense response, this reaction doesn’t occur in Carrizo and we are not able to assess resistance to X. citri in these plants. In Duncan grapefruit, our efforts to transform constructs with AvrGf1 and AvrGf2 transgenes, where an inducible hypersensitive response is expected, have led to the isolation of seven putative transformants. These were sequenced to determine whether the transgene construct was intact. We found that all seven had deletions in the area of the transgene comprising parts of the promoter region and Avr gene. We believe our difficulty in obtaining transgenic grapefruit is arising either because the construct may have a tendency to recombine in Agrobacterium or during the transformation process, or the promoter may be leaky at some point during the transformation process, even though we have shown that it is tightly regulated in transient assays in leaves. Therefore, we are taking further steps to assess the stability and background expression of the construct. To investigate construct stability, transgenic tobacco plants (Nicotiana tabacum) resulting from the constructs pCAMBIA2201, pCAMBIA2201:NosT:Bs3super::avrGF2, and pCAMBIA2201:NosT:Bs34box::avrGF2 have been generated to determine whether the constructs are stable in another system. If construct sequence optimization is necessary, it will be easier in the tobacco system. We are also carefully assessing the potential for background expression of the construct. Four Carrizo transgenics containing the 14EBE construct fused to GUS were obtained, and these will be verified for the presence of the intact transgene by PCR, followed by GUS staining to determine whether there is any unexpected expression in any tissues other than leaves. In addition, N. tabacum and N. benthamiana transgenic plants carrying the 14 EBE construct fused to GUS will be stained to determine whether GUS is expressed anywhere in the plant. If one of the added EBEs produces unwanted background expression, we will be better able to determine which one is problematic in this system. We will also attempt to transform Duncan grapefruit with a construct containing the Bs3 promoter without any added EBEs, fused to an Avr gene or to GUS, to assess whether the unaltered promoter produces any background expression. Work also continues in the tomato model system, where one transgenic line carrying the disease resistance construct showed a reduction in symptoms in initial tests. T2 plants are being generated for further study.
Objective 1: Generate functional EFR variants (EFR+) recognizing both elf18-Xac and elf18-CLas. In order to perform screening on complex EFR mutant libraries required to discover mutants which respond to elf18-CLas ,we have been developing a FACS-based screen. To this end we have generated a number of reporter lines (using GFP) in both suspension cultures and transgenic Arabidopsis plants. The reporter lines are driven by the FRK1, WRKY30 and PER4 promoters. We have tested two PER4p:GFP cell suspension lines for responsiveness to elf18, and both of these give clear induction of the reporter gene following treatment. However, when protoplasts were produced of these lines, elf18 responsiveness was no longer observed. We are currently retesting these lines to ensure the buffer conditions and EFR expression is correct. In addition, plant and cell suspension lines transformed with FRK1p:GFP and WRKY30p:GFP are also in the process of being tested. In addition to the mutagenesis approach, we screened the Nordborg collection of Arabidopsis ecotypes for sensitivity to elf18-CLas or reciprocal chimeric peptides of elf18-Ecoli and elf18-CLas. Of this collection, none show ROS in response to either elf18-CLas or the chimeric peptides. We did observed one line (Se-0) which had enhanced response to the CLas-Ecoli-elf18 chimera. This chimera has some activity in Col-0, but only at high concentrations. Further testing of this ecotype revealed that it also enhanced ROS to elf18-Ecoli and to flg22, indicating that it was not a variant of EFR which was causing the enhanced ROS. Indeed the sequence of EFR from Se-0 contains no non-synonymous SNPs. We have been also investigating the possibility of targeting other PAMPs. To this end we conducted a bioinformatic comparison of known PAMPs with those in C. Liberibacter asiaticus. From these search we identified CSP22 (Felix & Boller, JBC 2003, 278:6201) as a potential candidate, since it is conserved in the sequence required for recognition. We are currently waiting for delivery of the CLas-CSP22 peptide to test. Objective 2. Generate functional XA21-EFR chimera (XA21-EFRchim) recognizing axYS22-Xac. These constructs have been constructed and tested and a manuscript is under revision. Objective 3: Generate transgenic citrus plants expressing both EFR+ and XA21-EFRchim. Transformation experiments are ongoing; to date, a total of 5,781 ‘Duncan’ grapefruit and 956 sweet orange segments have been collectively transformed with the constructs EFR, EFR-XA21, EFR-XA21-EFRchim and pCAMBIA2201 (empty vector control). A total of 580 and 219 grapefruit and sweet orange shoots, respectively, were transferred to rooting media. These shoots were first analyzed histochemically for GUS expression. The results show that collectively 6 grapefruit shoots were GUS positive with the constructs EFR-XA21, EFR-XA21-EFRchim and pCAMBIA2201 and 1 sweet orange shoot GUS positive with the construct EFR-XA21-EFRchim. Other grapefruit shoots (47) collectively stained partially (chimeric) for GUS with the constructs EFR, EFR-XA21, EFR-XA21-EFRchim and pCAMBIA2201, while 5 sweet orange shoots stained chimeric for GUS with the constructs EFR, EFR-XA21 and EFR-XA21-EFRchim. Chimeric shoots were those segments with less than 85% of blue staining.
Evaluation of existing standard and non-standard cultivars (‘Hamlin’, ‘Temple’, ‘Fallglo’, ‘Sugar Belle’, ‘Tango’, and ‘Ruby Red’) for HLB resistance/tolerance is complete. In August 2010, the plants were established at Pico’s farm in Ft. Pierce Fl. Data on the growth rate, disease severity, and Candidatus Liberibacter asiaticus (CLas) titer levels have been collected since April 2012. All trees exhibited symptoms of HLB and tested positive for CLas. During the 4-year period, there were significant differences in disease severity, stem diameter, and CLas levels among the varieties. ‘Fallglo’ had the lowest incidence of HLB symptoms, whereas ‘Ruby Red’ had the highest incidence. ‘Ruby Red’ also appears to be in significant decline. The highest CLas titer levels were observed in November, December, and January with ‘Sugar Belle’ and ‘Tango’ had the highest titer levels while ‘Fallglo’ and ‘Temple’ had the lowest. Despite the high titer levels found in ‘SugarBelle’, it had the greatest overall increase in diameter and was the healthiest in overall appearance. These results indicate that compared to ‘Hamlin’, ‘Fallglo’ and ‘Temple’ appear to display field resistance to HLB while ‘SugarBelle’ appears to have significant tolerance. Imidacloprid was applied quarterly to a subset of trees and significantly increased stem diameter compared to the non-treated trees but did not have a significant effect on tree height, disease severity, or CLas titer levels. Progress has been made on the antibiotic treatment of HLB infected bud-wood. Bud-wood of nine HLB symptomatic varieties, 3 fairly resistant (‘Temple’, GnarlyGlo’, and ‘Nova’) 3 tolerant (‘Jackson’, FF-5-51-2, and Ftp 6-17-48), and 3 susceptible (‘Flame’, ‘Valencia’, and ‘Murcott’). In November 2013 and May 2014, HLB positive bud-wood was treated with various concentrations of penicillin and streptomycin and grafted on sour orange rootstock. In August 2014, standard growth measurements (stem diameter and height), disease severity were evaluated and leaves were sampled for qPCR analysis. Evaluations and sampling will continue on quarterly basis. Development of periclinal chimeras with resistant vascular tissue from Poncirus and remaining layers from sweet orange is currently underway. One hundred and fifty etiolated seedlings of the trifoliate ‘Rubidoux’ and the sweet orange ‘Hamlin’ have been approach grafted together. Generation of new chimeras has been difficult. Several adventitious buds have emerged from the treated graft region, and one appears to be a chimera. The newly emerged plants will be tested using LC/MS to determine the origin of the three layers. To increase the success rate, additional plants will be grafted over the next twelve months. In October 2013, 34 unique genotypes (USDA hybrids) some of which appear to have tolerance to HLB, and 16 standard commercial varieties were exposed to an ACP no-choice feeding trial and have been transferred to the field at Ft. Pierce Fl. Standard growth measurements and disease ratings were initiated in July 2014 and will continue on a monthly basis. In September 2014 there were significant differences in trunk diameter (p<0.0001). At this time there were no significant differences in disease symptoms. However, it is still too early to assess for HLB resistance/tolerance. Three leaves were randomly samples and CLas titer levels will be quantified using qPCR. LC-MS assessment of potential HLB resistant biomarkers in Citrus and Citrus relatives is being explored. A method for the rapid identification of potential sources of HLB resistance is also being developed. This project involves the screening of citrus seedlings at the 3 to 5 leaf stage, or very small micrografted trees, that are exposed to HLB infect ACP feeding. CLas titer levels, using real time PCR, are evaluated at 3, 6, and 9 weeks Seedlings of Hamlin and Dancy show early CLas proliferation and systemic movement. Only very low levels of CLas have been observed in Carrizo.
The work in the Core Citrus Transformation Facility (CCTF) continued without interruption. Co-incubation experiments using different type of explants and Agrobacterium strains are still being done on a weekly basis resulting in production of more transgenic plants. Three new orders were received during the last three months although most of facility’s productivity came as result of work on older orders. Significant effort was invested into production of rootstock plants carrying the NPR1 gene requested by the CRDF. About 850 shoots were tested in the primary PCR with 135 of them being positive. Forty eight of these shoots died either before or after grafting, and 27 were negative in the second PCR. The other 60 positive shoots were grafted. Out of those, 27 were moved to pots. Presently, there are 29 plants in the greenhouse that are 6-10 inches tall. In about six weeks many of those plants will reach the size when they could be cut into nodal explants for propagation. In the period covered by this report, CCTF produced plants for the following orders: pNPR1-30 plants, pNPR1-G-five plants, pX4- two plants, pELP3-G-one plant, pELP4-G- one plant, pMG105- eight plants, pPR2-one plant, pHGJ2- one plant, pHGJ8-one plant, pW14- two plants, pMED16-four plants, pN7-three plants, pN18- four plants. Out of total of 63 transgenic plants, 29 were Carrizo citrange, three were Swingle citrumelo, one was C. macrophylla, one was Mexican lime, one was Valencia orange, and 28 were Duncan grapefruit. Within previous year the efficiency of transformation and the ability of explants to regenerate shoots are little lower than they were in previous years. The reason for this is probably the low quality of seedling explants which are starting material in our experiments. Seeds used for production of Duncan grapefruit, Valencia orange, and Hamlin orange seedlings that are cut into explants are obtained from fruit harvested on CREC property where HLB is widespread. Fruit have unhealthy appearance; seeds are smaller and have altered color. Seed vivipary which occurs in older fruit of Duncan appears four months earlier than it used to. This is a strong indicator of disrupted hormonal balance within the fruit which may contribute to changes we noticed. For this reason, we intend to change the way fruit are acquired. Duncan grapefruit will be picked from CREC property only between September and January. Depending on the quality of fruit this season CCTF may start getting Duncan fruit from the outside source even before January. Fruits of sweet oranges will be acquired from the outside sources throughout the whole season and CCTF will require assistance for this.
The main accomplishments during this quarter: 1) We were continuing to infect and transform mature tissues of of citrus using Agrobacterium with the shoot enhancing genes we constructed. The explants used were greenhouse grown Washington Navel, Pineapple and Valencia. More calli formed than with the regular vectors. However, because the numbers of calli produced were relatively small, rates of shoot regeneration between the control vector and transformation enhancing vectors had not been compared. We were preparing a large number of adult explants for future infection experiments. We also started to use a number of techniques to reduce the contamination problems and a large number of explants of adult tissues for infection. 2) We were characterizing the enhancement of transformation efficiency of juvenile tissues of citrus using our regenreation enhancing genes in detail and also verified some of the results obtained previously. 3) Verification experiment for the role of an endogenous plant hormone in citrus regeneration from juvenile tissues upon transformation was performed and some progress had been made. However, more time is needed to generate significant results. We hope this study could shed some lights on the role of that particular hormone in adult tissue generation after infection. If so, the experimental results may guide designs of additional gene constructs for enhancing adult tissue transformation.
The main accomplishments during this quarter: 1) We improved a sterilization technique used for greenhouse-grown mature/adult shoot tissues and the contamination problems have been significantly reduced. 2) We have infected mature/adult tissues of Valencia and Washington orange using our transformation enhancing genes (K and I genes). Our preliminary results show that the use of the K and I genes we developed lead to drastic increases in transformation efficiency of mature tissues when compared to the conventional Ti-plasmid vector containing no K or I gene. 3) We have observed that the transport of an endogenous plant hormone in explants plays an important role in shoot regeneration efficiency. We also observed that manipulating the transport of that hormone improves shoot regeneration and genetic transformation efficiency of juvenile citrus explants. We are currently testing the effects of the same manipulation on adult tissues of citrus. We hope that manipulation can further enhance transformation efficiency of adult citrus tissues. 4) We are writing one manuscript that reports the drastic enhancement of citrus transformation efficiency of juvenile tissues of citrus. We will work on the second one, the effects of the transport and its manipulation for an endogenous plant hormones in explant tissues, once the first one is submitted.
We have concluded our phase 1 search employing our recently developed bioinformatics tools PAGAL and SCALPEL that led to the identification of 3 potential citrus candidate proteins that could serve as replacements for the CecB lytic peptide domain of our previously described chimeric antimicrobial protein (CAP; Dandekar et al., 2012 PNAS 109(10): 3721-3725). Using the same tools we have further refined our search within these particular proteins to identify a smaller segment that was tested for antimicrobial activity after chemical synthesis of the protein candidates. The following citrus proteins were chemically synthesized a 22 aa version of HAT (CsHAT22; a 52 aa segment of this protein was previously identified) a 15 aa segment of ISS (CsISS15 ‘ a negative control) and 20 aa segment of PPC (CsPPC20). These proteins were used to test the efficacy of their antimicrobial activity using the following bacteria, Xanthomonas, Xylella, E.coli and Agrobacterium. Using the same search criteria we identified a 22 aa N-terminal segment of the 34 aa Cecropin B (CBNT22) protein and a 12 aa segment of cathaylecitin (CATH15), representing protein with known antimicrobial activity that could serve a positive control for our bioassays. CsHAT22 and CsPPC20 were able to inhibit bacterial growth at levels comparable to CBNT22 and CATH15, however, CsISS15 displayed no detectable antimicrobial activity (as expected). We have recently obtained Liberibacter crescens and will soon test the antimicrobial activity with this bacteria as a surrogate for CaLas the causative agent of HLB. We have designed 3 constructs 1) CsP14a with a secretion sequence and Flag tag, 2) CsP14a ‘ CecB (this is construct 1 expressed as a CAP with the original CecB and 3) CsP14a-CsHAT52 (the 52 aa version of the CsHAT protein from Citrus) for testing in CTV vectors system and in transgenic plants (tobacco and citrus rootstock). These three constructs have been successfully incorporated into CTV vectors and are being infected to develop plant materials that can be used for challenge with HLB. All three of the above constructs have been introduced into binary vectors and then incorporated into Agrobacterium strains and these are being used to transform tobacco and Carrizo rootstocks. The plant transformation process in underway and will culminate in the isolation of transgenic plants that can be tested for disease resistance efficacy.
The main accomplishments during this quarter: 1) We did three Agrobacterium infections using adult tissues of Washington Navel, Pineapple and Valencia from greenhouse-grown plants. With the K and I genes, we observed more calli formed than with the regular vectors, which is a positive sign. However, the numbers of calli produced are relatively small comparing to those of the juvenile explants. We started the step to regenerate shoots from calli we have already produced but no significant results can be reported at this time. 2) We have observed endogenous concentrations of a hormone may play a role in citrus regeneration efficiency from juvenile tissues. We have started additional experiments to verify that observation. If that is true, we will modify the gene cassettes we originally designed accordingly.
This is a continuing project to find economical approaches to citrus production in the presence of Huanglongbing (HLB). We are developing trees to be resistant or tolerant to the disease or to effectively repel the psyllid. First, we are attempting to identify genes that when expressed in citrus will control the greening bacterium or the psyllid. Secondly, we will express those genes in citrus. We are using two approaches. For the long term, these genes are being expressed in transgenic trees. However, because transgenic trees likely will not be available soon enough, we have developed the CTV vector as an interim approach to allow the industry to survive until resistant or tolerant trees are available. A major goal is to develop approaches that will allow young trees in the presence of HLB inoculum to grow to profitability. We also are using the CTV vector to express anti-HLB genes to treat trees in the field already infected with HLB. At this time we are continuing to screen possible peptide candidates in our psyllid containment room. We are now screening about 80 different genes or sequences for activity against HLB. We are starting to test the effect of two peptides or sequences in combination. We have developed methods to be able to screen genes faster. Finally, we have found a few peptides that protect plants under the high disease pressure in our containment room with large numbers of infected psyllids. We now are examine combinations of peptides for more activity. We recently examined all of the peptides constructs for stability. The earliest constructs have been in plants for about nine years. Almost all of the constructs still retain the peptide sequences. One of the peptides in the field test remained stable for four years. We now are examining the possibility of treating infected plants with antimicrobial peptides to allow them to recover from an HLB infection. We are beginning to work with a couple of teams of researchers from the University of California Davis and Riverside campuses to express bacterial genes thought to possibly control Las. We are screening a large number of transgenic plants for other labs. We have promising transgenic plants that are being rescreened to ensure efficacy against HLB. We have had a collaboration with Dr. Zhonglin Mou, Department of Microbiology and Cell Science in Gainesville, to test transgenic plants over-expressing plant defense genes. We have found that three different lines appear to be giving strong tolerance against HLB. We are propagating the plants for more extensive analysis.
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