The objective of this project was to discover citrus germplasm with resistance to the Asian citrus psyllid. Research during the project showed that germplasm in the following major groups of Rutaceae did not have any significant resistance to infestations of the Asian citrus psyllid (ACP): sweet oranges, citrons, pomelos, limes, lemons, sour oranges, papedas, mandarins and hybrids among these groups including grapefruit. However, within the trifoliate group, most (but not all) accessions of Poncirus trifoliata as well as a number of its hybrids (.Citroncirus) were shown to have natural resistance to ACP. Two types of resistance have been identified. One of these resistance types (antixenosis) greatly reduces infestation levels of the psyllid, a resistance trait that may be related to differences in volatiles used by the psyllid to find and infest plants or the presence of a volatile that repels the psyllid. The other type of resistance (antibiosis) results in reduced longevity of psyllids, possibly related to the presence of toxic secondary plant metabolites. P. trifoliata can be crossed with Citrus species using traditional breeding methods, thus it may be possible to identify traits conferring resistance and to breed these directly into Citrus using traditional breeding or other methods. Additionally, volatiles imparting antixenosis may have value for managing or monitoring ACP. We have already discovered clear differences in the volatile profiles of susceptible and resistant plants. Poncirus trifoliata accessions (CRC code at the National Clonal Germplasm Repository, University of California, Riverside) identified as having antixenosis resistance to ACP: 838, 1498, 1717, 2552, 2554, 2861, 2862, 3151, 3206, 3207, 3209, 3210, 3212, 3213, 3215, 3217, 3218, 3219, 3330A, 3330B, 3338, 3411, 3412, 3484, 3485, 3486, 3547, 3548, 3549, 3571, 3586, 3588, 3882, 3888, 3938, 3939, 4006, 4007, 4009, 4017, and 4138 (41 of a total of 48 accessions evaluated). .Citroncirus (trifoliate hybrids) accessions with antixenosis resistance: 275, 1438, 1447, 1448, 1459, 1463, 2618, 3205, 3348, 3771, and 3881 (11 of a total of 34 accessions evaluated). Poncirus trifoliata and .Citroncirus accessions with antibiosis resistance: 275, 276, 1438, 1447, 1448, 1463, 3330B, 3348, 3881, 3889, and 3969 (11 of a total of 17 accessions evaluated). Two publications have thus far been published from this research project: Westbrook, C. J., D. G. Hall, E. W. Stover, Y. P. Duan and R. F. Lee. 2011. Colonization of Citrus and Citrus’related germplasm by Diaphorina citri (Hemiptera: Psyllidae). HortScience. 46 (7): 997-1005. Richardson, M. L., and D. G. Hall. 2013. Resistance of Poncirus and Citrus x Poncirus germplasm to the Asian citrus psyllid. Crop Science. 53: 183-188. Although this project is terminating, we are concluding antixenosis testing of a number of additional Poncirus and hybrid accessions, and an evaluation is being conducted on antibiosis of Poncirus to psyllid nymphs. Results of these experiments will be published. It is hoped that traits conferring antibiosis or antixenosis can be identified in the near future.
The transformation construct for expressing the FLT-antiNodT fusion protein in citrus is nearing completion. We encountered major difficulties cloning the FLT-antiNodT expression cassette into the pTLAB21 citrus transformation vector. The FLT-antiNodT cassette DNA appeared to be unstable in E. coli when cloned into pTLAB21, which stymied our cloning efforts for several months. The instability of the FLT-antiNodT cassette in pTLAB21 was surprising, since the FLT-antiNodT cassette was stable in E. coli when cloned into non-transformation vectors such as pBluescript. For reasons unknown, the FLT-antiNodT cassette was specifically unstable in pTLAB21. However, we serendipitously discovered that the inclusion of an additional segment of DNA next to the FLT-antiNodT cassette in pTLAB21 actually stabilized the FLT-antiNodT cassette in pTLAB21. This piece of DNA was derived from the original FLT-antiNodT cassette cloning vector, pBluescript. This additional piece of DNA should not cause a problem for transformation of citrus or expression of the FLT-antiNodT antibody in transgenic citrus. We are now in the process of sequencing and verifying the pTLAB21-FLT-antiNodT transformation vector. Once this process is complete, we will commence citrus transformations. We anticipate that transformations will begin within the next reporting period.
In the period between April and July of 2013, Citrus Core Transformation Facility (CCTF) experienced significant changes in personnel. These changes reflected negatively on the productivity and as result, the goal of 100 transgenic plants per quarter was not reached. The experimental efforts were directed towards orders that were placed within previous six months. Produced transgenic plants were all Duncan grapefruit. They belong to following orders: MED16-11 plants; X20-four plants; X16-three plants; X19-one plant; X4-one plant; Bi121-six plants; N5-31 plants; N7-nine plants; HGJ3-one plant; and W14-one plant. As opposed to a few previous quarters when the number of placed orders was unusually high, facility received three orders during this time. The ‘genes’ of interest in those vectors were: gMOD1, ELP3-CIV, and ELP4-CIV. All three orders require transformation of Duncan grapefruit plants. Actually, two more orders were placed but after initial attempts to mobilize binary vectors into appropriate Agrobacterium strains the quality of cultures was reviewed and client decided to withdraw them. Within the first year (14 months) of funding of this project, CCTF received 35 orders for production of transgenic plants. Altogether, about 430 plants were produced. They belong to 23 orders received within this funding period and eight orders from previous period. Transgenic plants that were requested from clients in new orders were mostly Duncan grapefruit, but there were some requests for Valencia oranges, Carrizo citranges, and Mexican limes. Ratio of produced plants reflects well the ratio of requested plants in placed orders. CCTF remained a reliable producer of transgenic material. High influx of orders speaks of the important role that CCTF plays in efforts directed towards disease tolerance improvement of Citrus cultivars. Researchers that have well developed ideas or initial encouraging data about the beneficial activity of certain genes know that CCTF can help them test their hypotheses by producing citrus plants that carry those genes in them. While waiting for transgenic material from CCTF, those research groups can direct their efforts towards development of appropriate challenge tests that will be performed on transgenic plants or work on other aspects of protection against citrus diseases.
This project was initiated May 1 2013 and we have focused our efforts on Activity 1. In this activity we are using a rational design strategy to develop and rapidly test individually the two components of a chimeric antimicrobial protein (CAP) based therapeutic that could provide broad spectrum resistance to young citrus plantings against a range of biotrophic bacterial pathogens (HLB and CBCD). Gene mining bioinformatics tools are being used to search existing citrus genomic DNA sequence information available in Phytosome (http://www.phytozome.net) to identify structurally relevant candidate citrus components that can be fashioned into a CAP molecule. The current and highly functional CAP design described by us (Dandekar et al., 2012 PNAS 109(10): 3721-3725) is being used as a scaffold/guide to search for the citrus components. Working with the developer of a recently described bioinformatic tool CLASP we have successfully searched using the active site architecture (3D shape) of the forst component HNE and have found a citrus PR14a sequence whose shape is similar. We have determined the similarity of the citrus protein shape to that of the tomato PR14 and grapevine PR14a proteins. We used the tomato sequence as the protein 3D structure has been determined and could thus be used to thread the citrus and grapevine protein sequences whose structure is yet to be determined. We have also been working on the finding the citrus DNA sequence to replace CecropinB component. We have identified several candidates and are evalusting the similarity in their structure with respect to the known structure of Cecropin B. We have begun the process of designing the vector system to express the citrus PR14a sequence that we have identified. This vector system will be used for the plant based expression system described in our Activity 2. Once designed and constructed will be expressed in Nicotiana benthamiana to produce active protein that can be tested for activity. We have also submitted an application to APHIS-PPQ for the movement of the recently cultured Leberibacter crescens (Lcre). Once we get permission then later this year we can test these proteins for their efficacy using the Lcre to evaluate growth and mortality.
We completed predictive computations on all proteins from the Citrus sinensis genome and prepared all web-pages needed for manual, expert-driven analysis. This preliminary website is available from here:
The site has been prepared and the contract has been signed to initiate construction.
The Core Citrus Transformation Facility (CCTF) continues to serve the community of researchers exploring ways to improve Citrus plants and make them tolerant/resistant to diseases. CCTF does its service by producing transgenic material. Within the last quarter, the CCTF facility worked on producing transgenic plants of the following combinations: produced the following transgenic citrus plants (transgene in parenthesis): Mexican lime (pHK vector); Duncan grapefruit (ELP3 gene); Duncan (MKK7 gene); Duncan plants (p7 gene); Duncan (p10 gene); Mexican lime and Hamlin (p33 gene); Duncan (SUC-CitNPR1 gene); Duncan (pWG19-5 vector); Duncan plants (pWG20-7 vector); Duncan (pWG21-1 vector); Duncan (pWG22-1 vector); Duncan (pWG24-13 vector); and Duncan (pWG25-13 vector). All of these plants are for researchers funded by CRDF in the battle against HLB and canker.
McTeer trial – (3.5-year old SugarBelle trees on 15 rootstocks, nearly 100% HLB infected as of September (2011)- remediation program initiated in January by application of southern pine biochar and Harrell’s UF mix slow release fertilizer): Slow release fertilizer was applied to all trees in January. Unlike sweet oranges, HLB-impacted SugarBelle fruit does not drop from the tree, and the impacted crop is still hanging on the trees, months after the typical harvest date. Rootstock effects on longer-term tree health are being monitored, trees on Orange #19 still look the best at present. St. Helena trial (20 acre trial of more than 70 rootstocks, Vernia and Valquarius sweet orange scions, 12 acres of 5.0 year old trees, Harrell’s UF mix slow release fertilizer and daily irrigation). CREC scouts assessed HLB again in March. HLB is spreading much more quickly in the zone with the traditional tree spacing (15×25); whereas it was very low in the highest density spacing (9×20). Big differences continued in HLB infection rates per rootstock, with the control commercial rootstocks continuing to show the highest infection rates, with most >80% infected. Sour orange-like hybrids (now 3.5 years old) are growing off very well with lower than expected HLB infection frequencies (mostly Pummelo x Shekwasha mandarin hybrids). Harrell’s UF mix was applied to all trees in January, and TigerSul was purchased for application in the next quarter (to avoid minor zinc deficiency observed last summer). Yield and fruit quality data collection was completed in March; statistical analysis underway. Greenhouse Experiments – Rootstock liners (test rootstocks and controls) were moved into the HLB-greenhouse and grafting with HLB-infected Valencia budsticks was completed for the rootstock comparison experiment (trees growing on slow release fertilizer). Liners of rootstock Orange #15 for the individual nutrient experiment were moved to the HLB-greenhouse – grafting will begin this quarter. Protection of seed source trees: The release of new and improved rootstocks to the Florida Industry will require a large and stable source of viable nucellar seeds for our nurseries. Since seed source trees will be growing in the HLB environment, such trees should be protected from HLB. 1. Agrobacterium mediated transformation to produce transgenic tetraploid Orange 16 plants containing the Snowdrop Lectin insecticidal gene (GNA) have been conducted. In addition, we have produced several Orange 16 plants containing the GNA gene (snowdrop lectin) fused with a Tobacco PR1b signal peptide for improved extracellular secretion of the GNA protein by plant cells and the GNA gene fused with a HDEL C-terminal extension for retention of the GNA protein in the endoplasmic reticulum. 2. Transgenic Carrizo containing a construct containing the Snowdrop Lectin insecticidal gene stacked with the antimicrobial gene CEMA (AMP) have been produced and are being propagated for analysis.
Progress with the rapid flowering system (pvc pipe scaffolding system) in the greenhouse: Several of the transgenic plants have reached the top of the scaffold, and the apical stems have been trained to grow down (expected to encourage early flowering). The goal is to reduce juvenility by several years to accelerate flowering and fruiting of the transgenic plants. Another rootstock with strong potential to influence juvenility was identified (Nova+HBP x sour orange + Flying Dragon). Seeds have been planted. Experiments to efficiently stack promising transgenes are underway. Experiments to efficiently stack promising transgenes are underway. Transgenic sweet orange plants containing a construct with CEME gene (AMP) stacked with the NPR1 (SAR inducer) gene have been evaluated. We have recovered 10 transgenic lines that contain both genes incorporated into the genome. We have also transformed our newly released sweet orange somaclone OLL#8 with this construct. Also, constructs containing the AttacinE gene stacked with the NPR1 gene and the CEMA gene stacked with the NPR1 gene have been produced, and transformation of OLL#8 and Valencia Sweet Orange is currently underway. Correlation of transgene expression with disease resistance response: Western blot analysis for plants containing LIMA and GNA are nearly completed, data is showing a strong correlation between transgene expression and desired phenotype. This supports the dogma that fairly large populations of transgenic plants are necessary (for each transgene/cultivar) to obtain adequate transgene expression while maintaining cultivar integrity. Improved transformation methodology (for seedless or recalcitrant cultivars, and eventually marker-free or ‘all plant’ consumer-friendly transformation): 1. In efforts to reduce transgene mediated metabolic load on the plant, we have transformed Hamlin suspension cultures with constructs containing our reporter gene (grape anthocyanin gene) driven by either an embryo-specific Carrot DC3 promoter or an embryo-specific Arabidopsis At2S2 promoter. It is expected that plants obtained from these constructs will not produce the reporter protein once a transgenic plant has been selected. Currently putatively transgenic embryos have germinated and are being grown to size for analysis. 2. The binary vector for an inducible cre-lox based marker free selection is under construction. We anticipate transformation experiments with this vector in the following quarter. Targeted transgene expression: ‘ additional transgenic plants of Duncan, Carrizo, Pineapple, Hamlin, and Valencia (produced with Agrobacterium-mediated transformation) containing the LIMA gene (AMP) controlled by AtSUS2 promoter (phloem specific) have been propagated by micrografting. Plant characterization and molecular analysis on these plants will begin the next quarter. In greenhouse evaluation (Southern Gardens w/ Mike Irey) of transgenic plants exposed to HLB positive psyllids, we observed several transgenic LIMA and NPR1 lines driven by a phloem specific AtSUC2 phloem-limited promoter to be HLB tolerant. Most of these lines were negative to qPCR after 2 years of evaluation and did not demonstrate visible disease symptoms.
Several anti-NodT scFv single-chain antibodies are now in hand. The anti-NodT antibody with the highest affinity for the target 30 amino-acid peptide has been selected and has been expressed successfully in E. coli. The plant gene expression construct consisting of the 35S Cauliflower Mosaic Virus promoter driving expression of a fusion of the flowering locus T protein with best-performing anti-NodT scFv protein has been produced. We are now in the process of transferring this 35S::FLT-scFv expression cassette into the pTLAB citrus transformation construct. We have been working on this construct for several months during January – April 2013, but still have not completed this step, which has proven to be unexpectedly difficult. We anticipate that we will be able to complete this step during the next quarter, using some alternate techniques. Once the construct is completed, production of transgenic plants will begin.
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 75 different genes or sequences for activity against HLB. We are starting to test the effect of two peptides or sequences in combination. We are attempting to develop methods to be able to screen genes faster. We are also working with other groups to screen possible compounds against psyllids on citrus. Several of these constructs use RNAi approaches to control psyllids. Preliminary results suggest that the RNAi approach against psyllids will work. We also continue to screen transgenic plants for other labs.
This is a joint project between CREC and USDA, Fort Pierce. The objective of this project is to find poncirus hybrids that exist now that are sufficiently tolerant and of sufficient horticultural and juice quality to be used now for new planting in the presence of high levels of Huanglongbing (HLB) inoculum. We believe there is a good chance that there mature budwood exists with these properties that could be available immediately for new plantings. Although these trees are not likely to be equal in juice and horticultural qualities of the susceptible varieties of sweet oranges grown in Florida, with their tolerance to HLB they could be an acceptable crutch until better trees are developed. We surveyed the trees at the Whitney field station and found 5 lines that we thought could be acceptable for juice. Those have been propagated and are being screened for tolerance and horticultural properties. The hybrid plants are being incubated in the psyllid containment room to allow multiple psyllids to inoculate the plants with HLB. So far the chosen hybrids appear to be tolerant.
Function of individual X. citri transcription activator like effectors (TALEs): The activity and specificity of specific X. citri TALE proteins PthA1-4 have been tested using two different approaches. In one approach, activity was tested transiently using a reporter assay in Duncan grapefruit leaves. In these assays, plants were co-inoculated with a reporter construct consisting of a 14 TALE binding element (EBE) version of the Bs3 promoter driving the GUSi reporter gene together with Agrobacterium containing individual pthA genes or combinations of genes. Our results from these studies show that on its own Xc PthA4 is the most effective activator of gene expression, however co-inoculation with other individual proteins increases expression. We also used a second approach in which stable transgenic Nicotiana benthiamiana plants containing a 4 EBE promoter:GUS construct were inoculated with individual citrus TALEs introduced via X. campestris pv. campestris. We then quantified activity using GUS leaf disc staining and fluorescence MUG assays. In both assays, we could observe that individual TALEs did trigger expression of the GUS gene, so long as the corresponding EBE was present. This system also permitted us to examine the activity of pthA genes from a range of strains, including the sequenced Brazilian A 306 strain, A44 from Argentina, a typical A strain from Miami, an unusual strain isolated from Etrog in Florida,and a C strain designated #93 Brazil. TALEs from these strains triggered expression of the construct when matching EBEs were present. These data show that the promoter constructs are functioning as designed, with specificity for individual TALEs from a wide range of strains. We also now have a number of assays to evaluate TALE-promoter interactions to evaluate the roles of TALEs individually and in combination. Transformation and production of stable citrus lines: We have experienced difficulty in recovering functional stable transgenic citrus with from our transformation efforts. We have obtained many transformants, but to date we have not identified a line with a functional, intact transgene construct. In response, we are pursuing several alternative approaches to obtain stable transgenics, including further examination and testing of original constructs, preparation of new constructs in alternative vectors, additional controls, optimization of the transformation protocol, and new methods of transformation. We continue to regularly set up transformation experiments and analyze transformants by PCR, sequencing and pathogen testing. Given the successful function of constructs in transient reporter and disease resistance assays and the success of stable transformation with other constructs, we expect to recover functioning stable transformants through continued optimization of the transformation process.
The goals of our integrated core program are to develop and evaluate new citrus scion and rootstock cultivars suitable for California conditions. Research on citrus scion breeding continued on schedule. An important development is addition of Dr. Soon Park to the breeding team as the lead scion breeder, replacing Tim Williams who has retired but remains active as a volunteer. To obtain low-seeded cultivars we irradiated budwood of Robinson, Lee, and Sidi Aissa mandarins and Cocktail grapefruit, and then propagated trees at Lindcove and UCR. New pollinations during spring 2013 are still in progress and more than 900 flowers have been pollinated. Evaluation of existing hybrids identified 22 promising selections; of these, three grapefruit hybrids were submitted to CCPP for cleanup prior to multi-location evaluation. Selections evaluated in multi-location trials included low-seeded selections of Lisbon lemon, Encore, Nova, Clementine Oroval , Kinnow, Fremont and several hybrids. No new varieties were released. For the low-seeded lemon selection, among eight locations mean seed counts ranged from 0.4 to 2.9 seeds per fruit. Although most fruit had 0-2 seeds, some fruit had 8-10 at several locations. Additional data is needed to determine whether to release this selection. Trees at Lindcove will be screened to evaluate productivity and seed content when not cross-pollinated. Rootstock breeding activities included propagation of over 800 new hybrids to be field-planted in 2013. Additional crosses made in 2012 produced about 1000 seeds. Seedlings from 32 varieties were grown for a Phytophthora tolerance trial. New rootstock trials for Clementine and DaisySL were planted at Lindcove and for DaisySL at CVARS (Thermal). We completed evaluation of field trials of Fukumoto navel at Lindcove and Tango mandarin at Porterville. Packline data was collected on the Porterville Tango and Lindcove Fukumoto trials but has not yet been analyzed. Seeds of rootstock selections were collected to propagate trees for new rootstock trials for lemons and possibly one for navels. We completed two harvests (Oct. 16-17 and Dec. 16- 17, 2012) of the replicated trial initiated in 2006 to evaluate 12 lemon selections for the California desert. Yield, fruit packout and exterior fruit quality data was collected at each harvest and fruit of each selection from the first sample date were also evaluated for interior fruit quality measurements. Evaluations of fruit from 48 cultivars introduced from outside California and corresponding commercial standards were completed including 23 Satsuma mandarins, 8 Clementine and other early season mandarins, 2 grapefruit hybrids, 9 navel oranges, 4 mid- and late season mandarins and 2 commercial type lemons. Results of our evaluations of 19 new Satsuma introductions in comparison with two commercial standards will be published in the March/April 2013 issue of Citrograph. Fruit of scion cultivars under development and new introductions were available for growers to see and taste at the Lindcove Research and Extension Center field day (Dec. 13, 2012), World Ag Expo (Feb. 11-14, 2013), UCR Citrus Day (Jan. 31 2013) and the California Citrus Mutual Citrus Showcase (March 7, 2013).
We aim in this project to genetically manipulate defense signaling networks to produce citrus cultivars with enhanced disease resistance. Defense signaling networks have been well elucidated in the model plant Arabidopsis but not yet in citrus. Salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) are key hubs on the defense networks and are known to regulate broad-spectrum disease resistance. With a previous CRDF support, the PI’s laboratory has identified ten citrus genes with potential roles as positive SA regulators. Characterization of these genes indicate that Arabidopsis can be used not only as an excellent reference to guide the discovery of citrus defense genes and but also as a powerful tool to test function of citrus genes. This new project will significantly expand the scope of defense genes to be studied by examining the roles of negative SA regulators and genes affecting JA and ET-mediated pathways in regulating citrus defense. We have three specific objectives in this proposal: 1) identify SA negative regulators and genes affecting JA- and ET-mediated defense in citrus; 2) test function of citrus genes for their disease resistance by overexpression in Arabidopsis; and 3) produce and evaluate transgenic citrus with altered expression of defense genes for resistance to HLB and other diseases. Currently we have cloned 8 full-length genes in these categories in the entry vector pJET and two of the genes were further cloned to the binary vector pBIN19plusARS. Transformation of Arabidopsis and citrus plants will be simultaneously performed to obtained transgenic plants over-expressing the constructs for further analysis of plant disease resistance. In addition, we are continuing to generate and/or characterize transgenic citrus plants expressing the SA positive regulators, as proposed in the previous project, although the support of this previous project has already been terminated. A manuscript describing the cloning and characterization of the citrus NDR1 ortholog was recently under revision in the journal Frontiers in Plant Science.
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