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. With this support, currently we have cloned six full-length genes with potential roles regulating SA, ET, and/or JA pathways to the binary vector pBIN19plusARS and transferred the constructs to Agrobacteria. All six Agro strains were sent to co-PI Dr. Bowman’s lab to initiate citrus transformation. In the mean time, we started the process of transforming Arabidopsis to overexpress these genes and to test their defense function. T0 transformed seeds have been harvested for some constructs and will be screened for transgenic plants followed by disease resistance tests. We aim to clone at least 10 citrus genes for testing their effectiveness in conferring disease resistance to HLB and citrus canker diseases. Additional gene cloning is underway. In addition, we are continuing to characterize transgenic citrus plants expressing the SA positive regulators, as proposed in the previous project (#129), although the support of the project has already been terminated.
We have continued to propagate plants from the mapping population to provide materials for greenhouse phenotyping via graft inoculations, increasing both the number of individuals propagated as well as the number of replicates of each genotype. We cannot accommodate the entire population in replicated fashion because of greenhouse space limitations, so we are preparing to test subsets of individuals in the greenhouse to compare with phenotypes observed in the field. Selected plants from several individual hybrids will be graft inoculated using infected budwood sticks in early spring 2014, once we have come through the winter-induced dormancy of some of the trifoliate hybrids. We have quantified HLB symptom rankings for all trees in the field planting, this past October. In addition, we have sampled our preselected subset of monitor trees, and the frequency of CLas detections is increasing compared to previous samplings. We have found at least one tree to be PCR+ for almost all selections assayed, though some of these have very high Ct values indicating very low concentrations of CLas DNA in such trees. The pure trifoliate orange types remain essentially PCR-, while control sweet orange trees are quite obviously affected by symptoms, and they also had low Ct values, indicating substantial CLas population levels. The hybrids appear to be segregating for tolerance, though not into clear and distinct categories. Finally, leaf samples were collected from all individuals in the field trial. DNA was extracted from more than 840 individuals total for qPCR runs, which have been completed. These results currently are being summarized. The same trends we see in our monitoring subset data are seen in the total populations. A post-doc has been hired to begin work on the project in January 2014.
We have continued to propagate plants from the mapping population to provide materials for greenhouse phenotyping via graft inoculations, increasing both the number of individuals propagated as well as the number of replicates of each genotype. These trees are being grown to a size suitable for inoculation with HLB infected budwood in an approved greenhouse structure at the CREC. We cannot accommodate the entire population in replicated fashion because of greenhouse space limitations, so we are preparing to test subsets of individuals in the greenhouse to compare with phenotypes observed in the field. We have continued to monitor all plants in the field for symptoms of HLB. The site where these are being grown is under very high pressure and the trees are not regularly sprayed for psyllid control. Unfortunately, the soils at this site are high pH and do not support vigorous tree growth, they produce nutrient deficiency symptoms, and the trees are Citrus x Poncirus hybrids; therefore recognizing obvious HLB symptoms is difficult. However, management at the site has improved and the trees are looking better than previously. In mid-September it was becoming more obvious that HLB was impacting several of the selections. We selected a subset of trees to develop baseline qPCR values as we proceed to assess infection by CLas over time. DNA was extracted from these trees as well as positive and negative controls, and qPCR was run. We were able to detect CLas in some but not all of the trees. We plan to revisit and record observations on symptom development in the next month when symptoms should be most easily seen, to run qPCR again on our subset of trees, and then to look at qPCR Ct values in the entire collection. Our GoldenGate SNP assay platform has been used to genotype the entire population; the data produced have been preliminarily analyzed, prior to developing a high density genetic linkage map, as proposed.
In this proposal our objective is to find citrus versions for the two proteins that make up the functional components of a chimeric antimicrobial protein (CAP) previously described by us (Dandekar et al., 2012 PNAS 109(10): 3721-3725). We have successfully identified a suitable replacement for the first component, the human neutrophil elastase (HNE) that also serves as the surface binding component of CAP. Since HNE is a serine protease with elastase activity whose 3D structure has been determined we used the PDB database to find a suitable plant protein with the same 3D structure. Using the active site geometry of HNE is a consistent structural feature we focused on a set of 288 non-redundant plant derived proteins extracted from the PDB database to narrow our search criteria. The key feature of our search involved using CLASP to search for a match using the electrostatic properties and structural geometry of the three amino acids that make up the active site of HNE. We obtained a close match with the tomato PR14a protein. Using the tomato amino acid sequences we then searched for a similar citrus protein by searching through citrus genome information in Phytosome (http://www.phytozome.net). This was successful and we have identified a single protein that has the identical amino acid sequence in both Citrus sinensis (Cs) and Citrus clementina (Cc) genomes. We have focused the 165 amino acid P14a protein from Cs which we refer to as CsP14a. We have analyzed this sequence and have determined that it is a secreted protein and contains what appears to be a 25 amino acid signal sequence. We have utilized the 137 aa mature protein and successfully constructed two synthetic genes that encode this protein, one that just contains the coding region of CsP14a and the other is a chimeric version that contains the CsP14a coding region linked to the CecropinB (CecB) protein. We have included a signal peptide (22aa) that we have used before and know works really well at secreting proteins to the plant apoplast and xylem. This signal peptide has been added at the N-terminal of this protein and we have added a Flag Tag also at the N-terminal so that the protein can be easily detected and purified. The Flag tag will remain a part of the secreted protein after cleavage of the signal peptide. We are constructing two CaMV35S expression cassettes to express both these synthetic genes. We are using CLASP to identify a citrus replacement component for CecB. Since CB has no enzymatic activity, we could not use a well-constrained motif like an active site. We chose instead the structural motif Lys10, Lys11, Lys16, and Lys29 a unique feature of CecB. Our analysis has proved fruitful and we have identified a good plant candidate that has the same shape and that is highly conserved in plants.
Our accomplishments are: 1) Various young and mature citrus plants and also citrus seeds that are used for this project were purchased, planted and maintained in a greenhouse; 2) Sterile culture of citrus plant materials were established; 3) Construction of the proposed genes that should enhance shoot regeneration and embryogenesis has been started and is well underway.
A gene obtained from Dr. Mou that confers tolerance to canker has been transformed into mature Hamlin, Valencia, Pineapple, Ray Ruby, Carrizo, and Swingle, and shoots are regenerating. Unfortunately a number of PCR positive Pineapple shoots that were micro-grafted onto Carrizo rootstock and maintained in liquid media were lost due to an error in media preparation. This error has been corrected. Six shoots of micro-grafted Pineapple survived from this batch and an additional 33 Pineapple shoots have been micro-grafted onto Carrizo rootstock. Shoots regenerated from Carrizo and Swingle explants are being elongated in tissue culture prior to rooting. GUS assays for the reporter gene and additional molecular analyses will be conducted once the shoots are larger. Two constructs obtained from Dr. Wang were also used in transformation experiments of mature Valencia. Shoots were micro-grafted onto Carrizo rootstock and are still growing in liquid media prior to secondary grafting or have already been transferred to the soil. Molecular analyses will continue once the plants are larger. Molecular analyses of Hamlin, Valencia, Pineapple, and grapefruit scion transformed with marker genes are underway. Thus far, nine out of ten GUS or GFP positive plants have tested positive for the expression of the nptII transgene using the nptII immunostrip assay. The remaining ~40 transgenics will be tested using nptII immunostrips, nptII ELISAs. and Southern blotting. The first flower buds have formed on a Valencia transgenic event originally generated on 5/30/12, so it has been ~19 months for flowering to occur which agrees with Dr. Pena’s protocol. One of the limitations of the mature scion transformation protocol is the relatively slow process of bud break of the scion following grafting and the slow growth of the scion for explants. The double budding procedure and daily hormone applications to induce early bud break have significantly increased productively of the growth room. Buds now break one week after the grafting tape has been removed. We have observed significant differences in bud break of the scion on different rootstocks following hormone application. The photoperiod has been extended to 19 hours of light and 5 hours of dark to further increase the rate of vegetative growth and productivity of the growth room. A number of scientists were contacted to provide additional constructs and three scientists indicated they will provide constructs in the near future.
The transformation construct for expressing the FLT-antiNodT fusion protein in citrus has been completed. We encountered major difficulties cloning the FLT-antiNodT expression cassette into the pTLAB21 citrus transformation vector. The FLT-antiNodT cassette DNA was 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 will not cause a problem for transformation of citrus or expression of the FLT-antiNodT antibody in transgenic citrus. We have performed DNA sequencing of the pTLAB21-FLT-antiNodT transformation vector and have verified its identity, stability in E. coli, and sequence integrity. The pTLAB21-FLT-antiNodT transformation vector was sent to the Citrus Transformation Facility at the University of Florida Citrus Research and Education Center at Lake Alfred, FL. The pTLAB21-FLT-antiNodT transformation vector was introduced into Agrobacterium tumefaciens strain EHA101. PCR was used to verify that the pTLAB21-FLT-antiNodT transformation vector was intact in EH101. Transformation of ‘Duncan’ grapefruit was initiated in late October, 2013.
St. Helena trial (20 acre trial of more than 70 rootstocks, Vernia and Valquarius sweet orange scions, 12 acres of 5.5 year old trees, Harrell’s UF mix slow release fertilizer and daily irrigation). The trial was again scouted for HLB, new infection frequency data per rootstock will be presented at the December 12th Field Day. Greenhouse Experiments – we finally obtained the polymer coated individual nutrients (kindly provided by Brian Patterson of Florikam), and 10 trees per treatment of rootstock Orange #15 were treated as follows: 1) regular liquid only control 2) Harrell’s only control; 3) Harrell’s + Ammonium Sulfate (Florikote); 4) Harrell’s +Urea (Florikote); 5) Harrell’s + Super Triple Phosphate (Florikote); 6) Harrell’s + Potash (Florikote); 7) Harrell’s + Magnesium Sulfate (Florikote); 8) Harrell’s + Sodium Borate (Florikote); 9) Harrell’s + Iron Sulfate (Florikote); 10) Harrell’s + Manganese Sulfur (TigerSul); 12) Harrell’s + Zinc Sulfur (TigerSul); 13) Schumann TigerSul mix; 14) Schumann TigerSul mix + biochar; and 15) biochar. Grafting of these trees with large budsticks of HLB-infected Valencia sweet orange was completed. We will now flush out the infected Valencia budsticks and determine the response of the trees to the various nutrient treatments. Protecting Seed Source Trees: 1. Transgenic orange 16 tetrazyg plants transformed with GNA and P-GNA have been produced and have been micrografted into Carrizo rootstock for rapid growth and development. Transgenic plants have been moved to the greenhouse for growth and subsequent multiplication. 2. Transgenic Orange 4 plants containing the GNA transgene produced in the first quarter have been clonally multiplied in a mist bed.
1. The binary vector for an inducible cre-lox based marker free selection has been constructed containing a heat inducible excision system containing the cre gene driven by a Soybean heat shock gene promoter. Tobacco has been transformed with the construct and we are observing a 35-50% excision of the selectable marker gene in the regenerated plants. Citrus epicotyl explants will be transformed with the same construct as seeds become available this fall. Transgenic plants containing a putative Citrus sinensis (sweet orange) small heat-shock protein gene promoter are also being generated. 2. Hamlin and W. Murcott cells have been transformed with a binary vector containing Dual T-DNA borders for gene segregation and marker free transformation of citrus suspension cell. Our data indicates that the transformation vector is functional and able to incorporate both T-DNAs into the plant genome. Putatively transgenic somatic embryos have begun to germinate and will be tested by once the plants are large enough. Experiments are being conducted to confirm if negative selection pressure can differentiate between cells that contain the marker free T-DNA from the T-DNA containing the selectable positive/negative fusion marker cassette and if it can be removed from the citrus genome. 3. The citrus FT gene has been incorporated into Carrizo citrange. Numerous transgenic plants containing the Citrus FT stacked with the citrus AP1 have also been produced for testing. Plants are growing in the laboratory and will be tested for the presence of the gene when they reach suitable size. 4. PCR analysis of transgenic plants containing the NPR1 gene stacked with the CEME transgene have identified 7 lines that contain both transgenes. These transgenic lines are being grown in the greenhouse for cloning and testing. Agrobacterium mediated transformation to produce more lines will commence in the following quarter. 5. Targeting AMP expression in the phloem: we have produced additional transgenic plants using Agrobacterium-mediated transformation (Duncan, Carrizo, Pineapple, Hamlin, and Valencia) with theLIMA gene controlled by the phloem limited AtSUC2 promoter. Some transgenic lines from these cultivars have been propagated for further characterization and molecular analysis is underway. 6. Transgene expression and correlation to disease resistance response: ‘ Western analyses for LIMA and GAN transgenes is nearly completed – transgenic plants from independent transformation events show quite variable transgene expression as expected.
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. We have modified the CTV vector to produce higher levels of gene products to be screened. 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 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 are screening a large number of transgenic plants for other labs. We are beginning to work with a team of researchers from the University of California Davis and Riverside campuses to express bacterial genes thought to possibly control Las. Since we are testing about 80 genes for induction of resistance or tolerance to HLB in citrus, we changing our focus of building new constructs to controlling psyllids until we have more conclusion from the peptides under screen. 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.
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. At this time, all 5 hybrids continue to appear to be tolerant to HLB. The inoculated plants are growing in the greenhouse as we wait to determine the degree of disease symptoms in each line.
We completed the first step in the analysis of Asian Citrus Psyllid proteome and compiled a website with the results:
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 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 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 10 full-length genes in these categories in the entry vector pJET. Five of the genes were further cloned to the binary vector pBIN19plusARS and transferred to Agrobacteria. The Agro strains were sent to our collaborator Dr. Bowman’s lab to initiate citrus transformation. In the mean time, we started the process of transforming Arabidopsis to overexpress these genes and to test their defense function. T0 transformed seeds have been harvested for some constructs and will be screened for transgenic plants soon. In addition, we did extensive bioinformatics analysis to identify potential new citrus defense genes to clone and to test for their defense functions. A list of 15 genes has been generated and primers to amplify these genes have been designed. The cloning of these genes will be initiated shortly. 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 new FT construct,FMVcDNA27, containing an FT3 cDNA insert in the pCAMBIA 2201 vector has been made with a constitutive FMV promoter that proves to be as effective at transforming citrus and tobacco as the corresponding genomic construct that has been previously used. The FMVcDNA27 construct will be used to develop a chemically inducible system for the expression of this transgene. The inducible promoter systems from the Danforth Foundation mentioned in the previous quarterly report update was not used due to unforeseen issues; therefore the work will be continued using a transcription activator-like (TAL) effector system inducible by methoxyfenozide that will activate the naturally present FT3 gene in citrus. This promoter will utilize chemical-inducible ecdysone receptor-based expression. Research has also been conducted that looks into other endogenous plant chemically inducible promoters that are not turned on by chemicals in the media used to transform plants for use in controlling the FT3 gene expression. A manuscript comparing the behavior of the three genomic clones from citrus when overexpressed in tobacco has been completed and is undergoing further review before publication. The one year study of the in vivo tracking of FT1, FT2, and FT3 in various citrus trees differing in age and phenotype has been completed and gene expression levels have been compared in a month-to-month basis using the comparative CT method from qualitative Real Time PCR. The results show a promising patter that could potentially clarify the flowering pathway and the physiological effects of these three genes as it relates to the induction of flowering. The data is being currently analyzed for statistical significance and will be cross-examined using a higher concentration of DNA to verify the results. SDS pages and western blots have been done with the synthesized FT3 protein in order to identify if the HA tag and antibody will be an effective method to test for the presence of the protein when applied to plant tissue. The synthesized protein showed high specificity the HA-antibody and therefore this method will be used to assess the presence of the FT3 synthetic protein in further studies. The same procedure was performed with the Arabidopsis FT antibody and endogenous FT in citrus, tobacco and Arabidopsis as to determine if this will be an appropriate assay for the detection of FT3 synthesized protein. Another experiment involving FT3 transgenic tobacco and the effect of plant hormones Ethylene and Gibberellin (GA) as well as a GA pathway inhibitor chemical Paclobutrazol is underway to determine an effective way of preventing precautious flowering of citrus FT3 transgenic plants in tissue culture stages.
The objectives of this project are: 1) to generate transcriptome profiles of both susceptible and resistant citrus responding to HLB infection using RNA-Seq technology; 2) to identify key resistant genes from differentially expressed genes and gene clusters between the HLB-susceptible and HLB-resistant plants via intensive bioinformatics and other experimental verifications such as RT-PCR; and 3) to create transgenic citrus cultivars with new constructs containing the resistant gene(s). The first group of 5 samples for RNA-Seq, including resistant/tolerant vs. susceptible plants are complete. The second group of 10 samples has been sequenced, and the data is subjected for detailed analysis. A couple of resistance genes are constructed for genetic engineering. We mapped the RNA-Seq data to a reference genome, C. clementina using the bioinformatics program STAR. About 85% of the raw reads could be uniquely mapped. The transfrags of each library were assembled with cufflinks and merged with cuffmerg. 24,275 genes of the originally predicted genes had been found to be expressed and a total of 10,539 novel transfrags were identified with cufflinks, which were missing from the original reference genome annotation. Some of the NBS genes were found to be expressed. For C. clementine and C. sinensis, there were 118,381 and 214,858 mRNAs or ESTs deposited in GenBank and 93 out of 607 and 221 out of 484 NBS related genes match one or more ESTs respectively. The number of ESTs varied from 1 to 25. The expression abundance of each gene was measured by FPKM. The distribution curves of density of FPKM of 5 samples are very similar, indicating that the gene expression is similar and the quality of sequencing is high. We also performed the principal component (PC) analysis study on the expressions of five samples. The plot of the first PC against the second PC showed that R2017 and R20T18 clustered together (the resistance group) and R19T23, R19T24 and R20T24 clustered together (the susceptible group). This result showed that the gene expressions were significantly different in resistant vs. susceptible citrus. Using cuffdiff, a total of 821 genes were identified as difference expressed genes (DE genes) between the two groups using both p-value and an FDR threshold of 0.01. Among them, 306 genes are up-regulated and 515 are down-regulated in resistant citrus. Using the program iAssembler, a total of 53,981 uni-transfrags were obtained. Most of the assembled uni-transfrags should be novel genes, compared with the citrus reference genome. To reveal the differences in resistance, we also identified the exon variations (SNP/INDEL). A total of 612,618 SNP/INDELs were identified using the mpileup method employed in samtools. We focused on two types of mutations that could contribute to the resistance difference. The first type of mutation is a mutation in the genes of susceptible citrus leading to pseudogenes (Type1) and the other type of mutation is a mutation in resistant citrus genes that may gain a new function (Type2). Type1 mutations should have a homozygote mutation genotype in the susceptible citrus. We identified 146 candidate genes having Type1 mutations, which produced high impact variations such as frame shifts, splice site acceptors, splice site donors, a start lost, a stop gain or stop lost, and 3,578 genes with Type2 mutations. We expect that as the number of libraries being sequencing increases, the number of candidates will be reduced to a reasonable number allowing for further validation. We identified a few LRR-PKs genes for further comparative study based on the RNA-Seq data. The results indicated sequence variations of these genes in different varieties are indeed due to SNP/indels, and some of them were annotated as putative pseudogenes because of a truncation or insertion. Further verification is underway.
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