Objective 1: Transform citrus with constitutively active resistant proteins (R proteins) that will only be expressed in phloem cells. By restricting expression to phloem cells we hope to limit the negative impact on growth and development. Results: In addition to the SSI4 obtained from Arabidopsis thaliana var. Columbia genomic DNA, we created two new constructs (5-4) AtSUC2/SSI4 and AtSUC2/ssi4 mutant derived from Nossen genomic DNA (lines obtained from Dr. Klessig). Out of 49 ssi4 transgenics, 18 showed a reduced stature phenotype. However, none of the ssi4 expressing transgenics displayed a severe dwarf phenotype seen when expressed using their native promoter. We visited Dr. Orbovic’s laboratory at the UF CREC at Lake Alfred and resolved a technical problem regarding the PCR-based screening of citrus transformants. Dr. Orbovic has subsequently identified transgenic citrus for our two clones: AtSUC2/snc1 and AtSUC2/ssi4 mutants. Objective 2: Develop a method to elicit a robust plant defense response triggered by psyllid feeding. By further restricting expression of the R protein to the single cell that is pierced by the insect stylet, we anticipate that a defense can be mounted without a manifestation of a dwarf phenotype. The PAD4/ssi4 transgenics were difficult to obtain. We screened three times as many To seeds to obtain 18 plants. From these, 50% developed the “smaller stature” phenotype. Conversely, almost all transgenic plants containing SSI4 (wt) developed normally. Of the 33 transgenics with the PAD4/SNC1 construct, the majority showed no detrimental effect on growth. However, from the 37 PAD4/snc1 mutant transgenics, 10 were smaller than normal. We obtained 27 transgenics with the PAD4-reporter and analyzed GUS expression. Originally, the PAD4 promoter was selected based on literature reports of its phloem-specific expression and wound-inducibility upon the insect feeding. Our results indicated four basic groups of PAD4/GUS reporter expression in transgenic Arabidopsis leaves as follows: 1) phloem-specific expression with no wound induction; 2) universal expression with strong wound induction; 3) restricted expression with no wound induction; and 4) strong wound expression only. Conclusions: Our GUS analyses of two phloem-specific promoters (AtSUC2 and AtPAD4) revealed that transgenic plants do not maintain strict phloem-specific expression. Moreover, we found no PAD4 plants with the expected phenotype of wound-specific expression limited to phloem cells. However, substitution of the native promoters for ssi4 and snc1 genes with AtSUC2 and AtPAD4 did result in a significant reduction in the severity of the growth retardation phenotypes. Evaluation of more transformants will give us a better picture of the feasibility of utilizing these two promoters to obtain the desired expression patterns in citrus.
Obj 2 Employ qPCR to detect Liberibacter in experimental adult psyllid cohorts given a range of acquisition-access periods as immatures or adults. Pineapple citrus seedlings at 4-5 leaf stage will be used to study the transmission of HLB using (1) ACP reared on HLB positive citrus plants (hot colony), (2) field collected ACP from HLB positive citrus and (3) ACP reared on orange jasmine (cold colony). Experimental seedlings will be grown in plastic cones filled with Fafard 4 P soil media. Three seeds will be sown in each cone for a total of 60 cones. Upon germination, their number will be reduced to one per cone. Ten seedlings (replicates) will be used for each of the above three groups of psyllids and ten seedlings will be kept without psyllids. Each seedling will be isolated with 10 adults from the respective group within a ventilated plastic cylinder. Adults will be allowed to feed for 4 weeks and removed at that time or earlier when their progeny is in fifth instar and ready to become adult in order to separate the adults used to inoculate the seedlings from their progeny that developed on the seedling. At that time samples of initial cohort of 10 adults will be released on each seedling. Nymphal progeny (4-5 instars) and seedlings will be collected and processed using PCR for the presence of Liberibacter. The experiment will be conducted in an air-conditioned glass house maintained at 26 ‘C and 60-80% RH. Hopefully, seedlings will be able to withstand psyllid feeding pressure during the course of the experiment. Obj 3 Define the gross association of Liberibacter in thick sections. Develop a gross anatomical road map of Liberibacter accumulation in key organs, tissues, and cells. Obj 4 Using the resultant road map, elucidate, at the TEM level, where Liberibacter accumulates. The oral box (see Sept 2010), has become the center of attention because it is the terminal portal of the transmission cycle. Our exploration of the proliferation patterns of the bacteria in the deshelled abdomen still continues with FISH as our primary technique, but we must know if the bacterial titers in the abdomen are of secondary importance to the titers that can develop inside the oral box irrespective of them. We have formative ideas on the construction of a new model for transmission in this kind of insect vector/pathogen relationship, which is in strong contrast to models based on virus, from which scientists draw their initial methodical approaches. Our first Z-section library consists of 211 100nm thick plastic sections of an oral box of an infected adult under the transmission electron microscope. We have compiled most of them as needed into an animation (see Jan 2011). We’ve started on our second Z-section library. It uses the oral box of an uninfected adult from which much of the cellular material has been leached. This will allow us to accurately map, in three dimensions, the configuration of the salivary ducts and esophagus as they merge into the stylets for secretion and ingestion, respectively.
Once we have been able to establish at IVIA the procedures and conditions to transform mature Hamlin, Pineapple and Valencia sweet oranges, we are in conditions to transfer the basic protocols to Florida. We believe they can be reproduced with little or no modification at the new laboratory that is being set up at the CREC. As we have also developed a genetic transformation system for mature Carrizo citrange, we are now incorporating a construct of interest into this genotype. The second objective of our project was to develop genetic engineering strategies to improve citrus tree management. In this sense, we proposed to reduce endogenous gibberellin levels in transgenic rootstocks to make them dwarf or semidwarf. Such rootstocks could provide reduced size to non-transgenic scion varieties grafted into them. With this aim, we have incorporated a hairpin construct into Carrizo citrange to silence an endogenous GA20-oxidase gene and them reducing bioactive gibberellin levels in growing shoots. After Agrobacterium-mediated transformation, the explants regenerated abundant callus and showed prolific shoots formation. Around 70% of the explants regenerated shoots in the light step. At this moment there are several transgenic (PCR-positive) shoots micrografted in vitro. They are still pending of grafting on vigorous rootstocks in the greenhouse and Southern blot verification in coming weeks/months. New experiments will be run with this construct and fresh starting plant material within the next couple of months. For improving citrus tree management, we also proposed to over-express flowering-time genes in both the Carrizo citrange rootstock and the Pineapple sweet orange scion. This objective was initiated one year ago or so, and we have now at least ten independent transgenic lines of Pineapple sweet orange and Carrizo citrange expressing either FT or AP1 flowering-time genes already established in the greenhouse. We continue characterizing them in detail. The PI and his greenhouse/growth room manager, Josep Peris, travelled to Florida last March 20-27th to visit the CREC and supervise the last steps of the growth room construction before been finalized (by the end of April, we guess). We suggested to revise minor details to make the facility more reliable and helpful for operators. We also short-trained the tissue culture technicians in horticultural practices. We checked substrate, seed stock and plant nutrition issues with the manager Dr. Zapata.
Preparation of potato psyllid libraries for sequencing. During this time, we have collected 6 potato psyllid samples: uninfected guts, uninfected adults, uninfected larvae, infected guts, infected adults, and infected larvae. Based on the Trizol/RNeasy hybrid RNA extraction protocol we developed, we can extract 1 ug total RNA for Illumina sequencing library construction from: 80 Asian citrus psyllid larvae/100 Asian citrus psyllid adults or guts/250 Asian psyllid salivary glands or 20 potato psyllid adults/50 potato psyllid larvae/100 potato psyllid guts. mRNA isolation, cDNA synthesize and paired end library production. We have constructed 8 Illumina Paired end libraries for whole transcriptome sequencing from 6 potato psyllid samples (uninfected guts, uninfected adults, uninfected larvae, infected guts, infected adults, and infected larvae) and 2 Asian citrus psyllid samples (uninfected guts and infected adults). Illumina sequencing and assembly. We have received from NCGR the Illumina assembled sequences from 6 potato psyllid libraries and 2 Asian citrus psyllid libraries. Each library resulted in over 24M sequences and the total set of ~160M sequences for 6 potato psyllid libraries and of ~65M sequences for 2 Asian citrus psyllid libraries. The last two Asian citrus psyllid libraries are to improve coverage over initial 454 runs (429995 ESTs for uninfected guts and 233891 ESTs for infected adults). Taking together with ~100M sequences from other 4 Asian citrus psyllid libraries (infected guts, uninfected adults, uninfected larvae and infected larvae), we would be able to get a deep coverage of ACP whole transcriptome. Characterization of the psyllid transcriptome and Identification of midgut-specific genes. Annotation, qPCR analysis of ACP transcripts and biological validation of novel/tissue-specific ESTs/genes were carried out. Several psyllid genes have been selected for full length cloning, expression profiling, enzyme assay and/or metabolism analysis as potential targets for RNAi constructions, plant transformation and function identification. ACP sequence data from prior sequencing. The 454 reads from whole body and gut libraries of the Asian Citrus Psyllid have been submitted to Genbank and are available for download from http://www.sohomotera.org. This site also has a query interface to the annotated 454 unitrans and the annotated Hunter Sanger unitrans (unique transcripts). Both assemblies and annotatin were performed with the PAVE software, where the 454 annotation used the Aug2010 Invertebrate and full SwissProt and TrEMBL databases and the Hunter used the corresponding Feb2011 UniProt databases. We are currently updating both annotations to use the Mar2011 UniProt databases, where we are searching against the invertebrate, virus and bacteria databases separately so that the composition of the functional annotation can be easily deciphered. For example, the results from the Hunter annotation shows the following number of unique hits to each database (up to 25 hits are stored for each Unitrans for each database): 4294 SP-Invertebrate, 31112 TR-Invertebrate, 125 SP-Virus, 599 TR-Virus, 6122 SP-Bacteria, 13309 TR-Bacteria, 5 SP-All, 7 TR-All (the All hits do not include any of the species database hits).
Huanglongbing (HLB) and Citrus Bacterial Canker (CBC) present serious threats to the future success of citrus production in the US. Insertion of transgenes conferring resistance to these diseases or the HLB insect vector is a promising solution. Genes for antimicrobial peptides (AMPs) with diverse promoters have been used to generate numerous transformants of rootstock and scion genotypes. New promoters and/or transgenes are being regularly introduced with more than a thousand new transformation attempts on citrus epicotyl sections each week. Plants have progressed from the initial round of scion transformations and are now replicated and are being exposed to HLB, using graft inoculations and CLas infected psyllids in greenhouse and field environments. Challenge with HLB through exposure to infected ACP (in collaboration with D. Hall) is being conducted on a replicated set (8 plants of each) of 33 independent Hamlin transformants, 5 Valencia transformants, 4 midseason transformants, and 3 non-transformed controls. It is anticipated that statistical analysis of CLas levels and symptoms will permit identification of material with significant resistance, for further testing. A series of promoters has been tested with the GUS gene to see how effective they are. As expected, the three vascular-specific promoters show expression only in phloem and xylem, while other promoters show broad expression in tested tissues. Sucrose synthase promoter from Arabidopsis drives high GUS expression more consistently than citrus SS promoter or a phloem promoter from wheat dwarf virus. A ubiquitin promoter from potato drives unusually consistent and high GUS activity. Use of this promoter may reduce the number of independent transformants needed. CLas sequence data were used to target a transmembrane transporter (Duan collaboration),as a possible transgenic solution for HLB-resistance. Radiolabelled ATP is being used assess effect of identified peptides in E. coli expressing the CLas translocase. Collaboration with a USDA team in Albany, CA is: providing constructs with enhanced promoter activity, minimal IP conflicts, and reduced regulatory and consumer concerns; providing genes from citrus genomic data, from Carrizo citrange sequence generated using USDA funds, to permit transformation and resistance using citrus-only sequences; citrus-derived T-DNA border analogues have been shown to be effective in producing transgenic Carrizo and tobacco and are being tested in citrus scions. Anthocyanin production genes,give bright red shoots (Gray collaboration) and are being tested as a visual marker for transformation, as a component of a citrus-only transgenic system. Transgenes are being developed to suppress (using an RNAi strategy) a lectin-like protein produced in the phloem of HLB-infected citrus. It is possible that suppression of this protein may significantly reduce disease symptoms. High throughput evaluation of HLB resistance will require the ability to efficiently assess resistance in numerous plants. Graft-inoculation, controlled psyllid-inoculation, and ‘natural’ psyllid inoculation in the field are being compared. The first trial has been in the field for 27 months and a repeated trial has been in the field for 15 months. Leaf samples have been collected monthly and PCR analysis of CLas conducted.
As proposed, a transgenic test site has been prepared at the USDA/ARS USHRL Picos Farm in Ft. Pierce, where HLB and ACP are widespread. The first trees have been in place for more than fourteen months. Dr. Jude Grosser of UF has provided 300 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser has just planted an additional 89 tress including preinoculated trees of sweet orange on a complex tetraploid rootstock that appeared to confer HLB resistance in an earlier test. USHRL has a permit approved from APHIS to conduct field trials of their transgenic plants at this site, with several hundred transgenic rootstocks in place. An MTA is in place to permit planting of Texas A&M transgenics produced by Erik Mirkov. More than 120 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) have been propagated for a replicated trial in collaboration with Fred Gmitter of UF and are growing well in the greenhouse. These will be planted in the spring of 2011, and 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. An experimental attract/kill product, to disrupt citrus leaf miner (CLM) without disrupting ACP, was not effective last year. Our experience suggests CLM may significantly compromise tree growth where insecticides are avoided to permit ready transfer of Las by psyllids. CLM damage also compromises ability to view HLB symptoms. Several applications of Admire are being used to encourage an undamaged flush on transgenic trees. We are still learning how to grow trees for best assessment of HLB-resistance.
Work continued to determine the suitability of Rutaceae species for the development of the Asian citrus psyllid and as hosts for the various species of Candidatus Liberibacter. The psyllid successfully colonize and reproduce on Choisya ternata, C. arizonica, and Helietta baretta in no-choice tests, but reverted back to its preferred hosts, orange jasmine (Murraya paniculata) and curry leaf (Bergera koenigii) in choice tests. On Amyris madrensis, A. texana and Zanthoxylum fagara eggs were laid and hatched but no nymphal development beyond first instart was found. Psyllid reproduction did not occurred on Esenbeckia berlandieri, Ptelea trifoliata nor Casimiroa edulis, although adult survived on these species. Severinia buxifolia is a frequently grown ornamental on which psyllids feed and reproduce. As previously reported we were able to transmit Ca.Liberibacter asiaticus to it by grafting. Symptoms in S. buxifolia were very nondescript with only thickened leaves and some vein corking. Live bacterial populations were measured monthly in the plants using a newly devised qPCR method. Transmissions of Candidatus Liberibacter asiaticus were successfully done from S. buxifolia to both healthy sweet orange and to healthy S. buxifolia in two separate tests. To date we have a 50% transmission rate from S. buxifolia to sweet orange and a 72% rate from S. buxifolia to S. buxifolia. Data showed that the bacterial populations in S. buxifolia declined over time if not exposed to infective PCR positive psyllids. Generic PCR primers developed were successful in detecting all know species of Candidatus Liberibacter. In S. buxifolia the titer of the Ca. Liberibacter asiaticus began getting lower like that seen with M. paniculata. In testing these plants with the generic primers a good titer of Ca. Liberibacter was found. The identification of this bacterium is ongoing. Esenbeckia sp.was identified as qPCR positive after psyllid transmission tests making it a new host for Ca. Las. Ca. Liberibacter asiaticus infected periwinkle and dodder were found to have higher live bacterial levels than source citrus plants. These studies will determine if certain plants are good hosts of live bacteria. Attempts to transmit Ca. Liberibacter africanus with D. citri failed. The number of psyllids used, length of acquisition and inoculation periods had no effect. The psyllid colony were pre-tested for presence of the bacterium and were tested again after acquisition and after inoculation and all tested negative for the bacterium. Ca. Laf source plants had Cq values in the low 20’s. Dodder transmission of Ca. Laf from sweet orange to Choisya X Aztec Pearl, periwinkle, and sweet orange was obtained but not to Severinia buxifolia. Dodder was highly infected as was the sweet orange and periwinkle, but Choisya had lower bacterial levels. Two sets of general ITS primers and new specific Ca. Laf ITS primers (Postnikova 2011) were used to generate PCR products for cloning and sequencing. Five clones from Laf infected Choisya were confirmed to be Laf. Murraya paniculata was confirmed to be a host of Laf, by PCR, cloning and sequencing. Tests were initiated to dodder transmit Laf to Bergera koenigii and M. paniculata. C. ternata, Choisya X. Aztec Pearl, and Choisya ‘Sundance’ were inoculated with the B239 Taiwanese isolate of Las with psyllids and Choisya X Aztec Pearl with dodder. To date all PCR assays have been negative with psyllid transmission to C. ternata. Fortunella polyandra, Clausena harmandiana, and C. excavata were obtained for testing. Work to further test the susceptibility or resistance of the cultivar IAPAR73 continued using psyllid transmissions and low numbers of qPCR positive plants were obtained.
Rutaceae species are indigenous in North America. Work continued to determine the suitability of some of these plants for the development of the Asian citrus psyllid as hosts for the various species of Candidatus Liberibacter. Using no-choice and choice experiments the psyllid was successfully colonize and reproduce on Choisya ternata, C. arizonica, and Helietta baretta in no-choice tests, but reverted back to its preferred hosts, orange jasmine (Murraya paniculata) and curry leaf (Bergera koenigii) in choice tests. On other species (Amyris madrensis, A. texana, Zanthoxylum fagara) eggs were laid and hatched but no nymphal development beyond the first instart was found. No psyllid reproduction occurred on Esenbeckia berlandieri, Ptelea trifoliata nor Casimiroa edulis, although adult psyllids survived on these species for a few days. Severinia buxifolia is a frequently grown ornamental on which the psyllids feed and reproduce. As previously reported we were able to transmit Ca.Liberibacter asiaticus to it by grafting since it is graft compatible with citrus. Symptoms in S. buxifolia were very nondescript with only thickened leaves and some vein corking. Live bacterial populations were measured monthly in the plants using a newly devised qPCR method. Data showed that the bacterial populations in S. buxifolia declined over time once they were no longer inoculated with Ca. Liberibacter asiaticus positive psyllids. Generic PCR primers developed were successful in detecting all know species of Candidatus Liberibacter in PCR. In S. buxifolia the titer of the Ca. Liberibacter asiaticus began getting lower like that seen with M. paniculata. In testing these plants with the generic primers a good titer of Ca. Liberibacter was found. The identification of this bacterium is ongoing. Ca. Liberibacter asiaticus infected periwinkle and dodder were found to have higher live bacterial levels (as determined by newly devised qPCR) than source citrus plants. These studies will determine if certain plants are good hosts of live bacteria. Attempts to transmit Ca. Liberibacter africanus with D. citri failed. The number of psyllids used, length of acquisition and inoculation periods had no effect on transmission. The psyllids were pre-tested for presence of the bacterium and were tested again after acquisition and after inoculation and all tested negative for the bacterium. Source plants had Cq values in the low 20’s indicating high levels of Ca. Liberibacter africanus. Dodder transmission of Ca. Laf from sweet orange to Choisya X Aztec Pearl, periwinkle, and sweet orange was obtained but not to Severinia buxifolia. Dodder was highly infected as was the sweet orange and periwinkle, but Choisya had lower bacterial levels. Two sets of general ITS primers and new specific Ca. Laf ITS primers (Postnikova 2011) were used to generate PCR products for cloning and sequencing. Five clones from Laf infected Choisya were confirmed to be Laf. Murraya paniculata was confirmed to be a host of Laf, by PCR, cloning and sequencing. Tests were initiated to dodder transmit Laf to Bergera koenigii and M. paniculata. C. ternata, Choisya X. Aztec Pearl, and Choisya ‘Sundance’ were inoculated with the B239 Taiwanese isolate of Las with psyllids and Choisya X Aztec Pearl with dodder. To date all PCR assays have been negative with psyllid transmission to C. ternata. Fortunella polyandra, Clausena harmandiana, and C. excavata were obtained for testing. Work to further test the susceptibility or resistance of the cultivar IAPAR73 continued and PCR testing is underway.
Previous work in our lab showed that LAS concentrations are the highest in the seeds of LAS-infected fruit. Since then, our focus has been on: 1) developing a method to aseptically obtain an inoculum from the seeds and 2) implementing a method to estimate the viability of LAS cells extracted from the seeds. Initially, an inoculum was obtained from LAS seeds by bead-beating whole seeds with metal beads and combing the seed pulp with a basic liquid media to obtain a suspension. An equal amount of this suspension was added to different media treatments and the relative concentrations of LAS cells in each treatment were determined by quantitative PCR. However, qPCR of replicate treatments had high variability. Since this was most likely due to the non-homogenous nature of the suspension (still contained some visible seed particles), efforts were made to create a more homogenous inoculum. Future suspensions were filtered through a 100 ‘m filter to remove large particles. Subsequent qPCR analyses of replicate treatments were much less variable. Next, a method was implemented to estimate LAS cell viability in the inoculated treatments over time. Ethidium monoazide (EMA) was used in conjunction with qPCR to determine the percentage of viable LAS cells. EMA binds to non-viable DNA so that it is not amplified by qPCR. Initial cell viability is variable, but is on average about 5% from the seed inoculum. After one week of incubation in flask containing various media treatments, however, no viable cells could be detected. Previously, LAS bacterial inoculum was also placed in sterile microfluidic chambers for observation under the microscope. Bacteria were observed in the samples in the microfluidic chambers. After about 2 weeks, the contents of the microfluidic chambers were eluted. LAS concentrations were determined by qPCR. Some LAS DNA was detected in the channels, but it is unknown whether this DNA was viable or not because the EMA viability assay was not implemented at that time. Future work will try to assess the viability of cells in the microfluidic chambers.
In this second quarter, 400 plants of Valencia orange on Kuharske Carrizo rootstocks were grown from rooted cutting in 1-gal plastic nursery containers and kept in the greenhouse under natural light conditions at 17-25’C. The plants will be used to conduct three different experiments that will include infected and non infected plants as well as treated non treated plants. For the fist experiment we will apply therapeutic compounds to HLB infected and healthy plants that will enhance Systematic Acquired Resistance (SAR) response and counteract ethylene in early infected tissues. The therapeutic compounds that we will be using in this experiment are Benzothiadiazole (BTH), Actigard, Salicylic acid, .-Aminobutyric acid (BABA). We will use untreated plants as controls. This will equal 5 treatments x 2 disease status (healthy and infected) x 10 biological replicates = 100 trees. For the second experiment we will be looking the regulation of glucose transport on HLB infected and non infected plants using gibberellin (GA3), the cytokinin 6-benzyladenine, KNO3 and a combination of the GA3, 6 benzaladenine and KNO3. We will use untreated plants as controls. This experiment will include 5 treatments x 2 disease status (healthy and infected) x 10 biological replicates = 100 trees For the third experiment we will examine sucrose + atrazine at two different rates along with an untreated control. The treatments will be applied at two different times to HLB infected and healthy plants, after infected plants become PCR+ for HLB infection. This will equal 3 treatments x 2 (healthy and infected plants) x 2 application times x 10 biological replicates = 120 trees . Plants infection will be done on the 3rd week of April by grafting infected bark pieces which will be obtained from citrus infected trees from commercial orchards. HLB infection for the bark pieces will be confirmed by qPCR 3 months after infection.
In this second quarter, 400 plants of Valencia orange on Kuharske Carrizo rootstocks were grown from rooted cutting in 1-gal plastic nursery containers and kept in the greenhouse under natural light conditions at 17-25’C. The plants will be used to conduct three different experiments that will include infected and non infected plants as well as treated non treated plants. For the fist experiment we will apply therapeutic compounds to HLB infected and healthy plants that will enhance Systematic Acquired Resistance (SAR) response and counteract ethylene in early infected tissues. The therapeutic compounds that we will be using in this experiment are Benzothiadiazole (BTH), Actigard, Salicylic acid, .-Aminobutyric acid (BABA). We will use untreated plants as controls. This will equal 5 treatments x 2 disease status (healthy and infected) x 10 biological replicates = 100 trees. For the second experiment we will be looking the regulation of glucose transport on HLB infected and non infected plants using gibberellin (GA3), the cytokinin 6-benzyladenine, KNO3 and a combination of the GA3, 6 benzaladenine and KNO3. We will use untreated plants as controls. This experiment will include 5 treatments x 2 disease status (healthy and infected) x 10 biological replicates = 100 trees For the third experiment we will examine sucrose + atrazine at two different rates along with an untreated control. The treatments will be applied at two different times to HLB infected and healthy plants, after infected plants become PCR+ for HLB infection. This will equal 3 treatments x 2 (healthy and infected plants) x 2 application times x 10 biological replicates = 120 trees . Plants infection will be done on the 3rd week of April by grafting infected bark pieces which will be obtained from citrus infected trees from commercial orchards. HLB infection for the bark pieces will be confirmed by qPCR 3 months after infection.
This project is focused on understanding the phloem disruption response from infection with the HLB bacteria, Candidatus Liberibactor asiaticus (LAS). Phloem distruption leads to starvation of the roots and tree decline. Callose and phloem protein 2 plugging are present in about a 3 to 1 ratio in field plants. Some virulence factors of the bacteria may cause necrosis responses. In the third year we want to fully understand if both phloem plugging and direct virulence of the bacteria play a role in decline development or by only one of these factors. Grapefruit trees with PAPETALA3-IPTgp (isopentenyl transferase) showing elevated (1, 3)-.-glucanase expression (callose polymer forming enzyme) were subjected to LAS by graft or psyllid inoculation and grown in the greenhouse for over one year. By January 2011 all plants had some HLB symptoms, but intensity did vary. Phloem samples will be prepared and examined for plugging quantity and types. This will tell whether multiple copies of the gene did block callose formation and the plants declined due to other bacterial induced virulence or the multiple copies or the elevated (1, 3)-.-glucanase expression caused more callose formation. Transformation experiments were continued in efforts to produce plants that over-express the citrus ‘-1,3-glucanase gene, both with the constitutive 35S promoter (p35S ‘ BG-35T) and the Suc 2 phloem specific promoter (pSuc2-BG-35T). Approximately 40 transgenic plants (Valencia and precocious sweet orange Vernia) have been successfully micrografted to Carrizo and are growing well in the greenhouse. Continued protoplast transformation experiments attempting to transfer the ‘-1,3-glucanase gene resulted in the recovery of GFP-positive somatic embryos as follows: 331 of sweet orange OLL#20, 39 of sweet orange ‘Jin Cheng’, and 10 of ‘Valencia’. Approximately one third of these embryos are normal and healthy, and should allow for transgenic plant recovery. LAS causes severe symptoms in plant compared to the psyllid. We hypothesized that a number of pathogenicity/virulence related genes of the bacterium would be overexpressed in planta, compared to the psyllid. To test this hypothesis, quantitative real-time PCR assays using total RNA isolated from infected plants and psyllids were conducted. Gene specific primers were used to check the expression of 560 genes in LAS. Genes showing a differential expression of two fold or more in either the plant (167) or psyllid (108) were categorized into Clusters of Orthologous Groups of proteins functional categories. Those genes are being further confirmed using different batches of samples to guarantee its accuracy. Two potential virulence genes that were overexpressed in planta along with 10 more constructs of putative virulence genes are being generated. Differential expression of these selected genes were also evaluated in more or less susceptible citrus types infected with LAS. Two potential virulence related genes were then screened on Nicotiana benthamiana plants for symptom expression, using transient assays and showed significant effects on tobacco.
Continued efforts to improve transformation efficiency: ‘ Experiments to test or validate the enhancing effects of various chemicals for improvement of transformation efficiency in juvenile tissues continued. Results showed that the use of the antioxidant lipoic acid significantly improves transformation efficiency in Mexican lime, and a manuscript reporting this was accepted with revision for publication in PCTOC. Recovered transgenic Mexican lime trees 1.5 years after removal from tissue culture were girdled, which induced flowering about 4 months later; with complete flowering and fruit set in less than two years. Experiments to test this with commercial sweet oranges are underway. We continued with experiments to test the effects of various antibiotics / metabolites / herbicide on the transformation efficiency, including: kanamycin, hygromycin, mannose and phosphinothricin. Horticultural manipulations to reduce juvenility in commercial citrus: ‘ Working with Mr. Orie Lee and Faryna Harvesting, yield and fruit quality data was collected from the St. Helena project. Approximately 10 acres of trees planted 3.0 years ago include a juvenile Valencia budline (Valquarius) and precocious Vernia on more than 70 rootstocks. The best rootstocks identified to show positive affects on rapid tree growth with precocious bearing and good early fruit quality (higher lb. solids) were somatic hybrids Changsha mandarin+trifoliate orange 50-7, white grapefruit+trifoliate orange 50-7, and sour orange+Carrizo, and tetrazygs White#4 (Nova+HBPummelo x Succari sweet orange+Argentine trifoliate orange), Orange#13, Orange#14,Orange#18 and Orange#19 (Orange series all Nova+HBPummelo x Cleo+Argentine trifoliate orange). Trees on these rootstocks averaged more than a half-box of fruit per tree with juice brix values great than 11. These rootstocks will now be tested to determine if they can shorten juvenility in transgenic plants produced from juvenile explant. Transformation of precocious but commercially important sweet orange clones: ‘ Transgenic plants of precocious OLL and Vernia sweet oranges were successfully micrografted to Carrizo citrange or experimental Tetrazyg rootstocks and are growing well in the greenhouse. These will now be clonally propagated onto the rootstocks mentioned above for further study of early flowering and transgene expression. Horticultural manipulations on these plants will include the RES (Rapid Evaluation System) growth method plus girdling. Transformation with early-flowering genes: ‘Citrus has at least 3 FT genes. Cloning and characterization of all 3 (genomic clones) has been completed. We have put them into transformation vectors with a constitutive promoter and performed transformation experiments with Carrizo and Duncan. Although only a few transgenic plants were recovered, we know the constructs work because we had previously tested them in tobacco, where we recovered early flowering phenotypes, as well as some other phenotypic alterations. Our current theory is that expression with the 35S promoter is too strong in citrus (in some cases we got flowering directly on the initial explant!). We are currently testing a poplar FT with a weaker inducible promoter (a heat shock promoter shown to be inducible in Oregon); which will hopefully solve the promoter problem.
This is a project to find an interim control measure to allow the citrus industry to survive until resistant or tolerant trees are available. We are approaching this problem in three ways. First, we are attempting to find products that will control the greening bacterium in citrus trees. We have chosen initially to focus on antibacterial peptides because they represent one of the few choices available for this time frame. We also are testing some possible anti-psyllid genes. Second, we are developing virus vectors based on CTV to effectively express the antibacterial genes in trees in the field as an interim measure until transgenic trees are available. With effective antibacterial or antipsyllid genes, this will allow protection of young trees for perhaps the first ten years with only pre-HLB control measures. Third, we are examining the possibility of using the CTV vector to express antibacterial peptides to treat trees in the field that are already infected with HLB. With effective anti-Las genes, the vector should be able to prevent further multiplication and spread of the bacterium in infected trees and allow them to recover. We now are making good progress: ‘ We continue to screen potential genes for HLB control and are finding peptides that reduce disease symptoms and allow continued growth of infected trees. ‘ We have greatly improved our efficiency of screening . ‘ We are modifying the vector to express more than one anti-HLB gene. ‘ We are modifying the vector to allow addition of a second vector. ‘ We are preparing to put trees into the field for testing as soon as potential freezes are over. ‘ We continue to supply infected and healthy psyllids to the research community.
A major objective of this project is to develop an understanding of how the HLB bacterium (Las) interacts with citrus genotypes to cause disease. After finding that different citrus genotypes respond differently to Las from extremely sensitive (sweet orange and grapefruit) to tolerance with minor symptoms, we have focused on the one citrus genotype that is most resistant to citrus. Las is restricted to very low levels in Poncirus trifoliata. Most plants remain PCR negative, but a few have barely detectable levels of Las. We have found that under some conditions Las appears not to be able to move through poncirus. We have plants with lower living inoculum that is highly infected with Las, but sensitive sweet orange shoots grafted on top of the poncirus plants have not become infected. We are examining the value of using Poncirus rootstocks and interstocks to reduce or prevent spread of the disease in sweet orange or grapefruit. We have developed a containment plant growth room to examine natural infection of citrus trees by psyllid inoculation. We have made several significant observations: First, we have found that the time period between when plants first become exposed to infected psyllids and the time that new psyllids can acquire Las for those plants can be as little as 6 weeks. We now are focusing on when and how psyllids acquire Las from newly infected plants. This information is necessary of the epidemiology models for managing HLB. We also have developed methods to greatly speed up results of field tests for transgenic or other citrus trees or trees being protected by the CTV vector plus antibacterial or anti-psyllid genes. In order to interpret results of a field test, most control trees need to become diseased. Under natural field pressure in areas in which USDA APHIS will allow field tests, this level of infection could take 2-3 years. By allowing the trees to become adequately inoculated by infected psyllids in a containment facility, we can create the level of inoculation that would naturally occur in the field within 2-3 years in 2-5 months in the containment room, after which the trees are moved to the field test site. We have greatly optimized conditions to allow exposed plants to become rapidly inoculated by infected psyllids. We continue be a resource of healthy and infected psyllids and plants for other laboratories and we are building CTV expressed genes to control psyllids or Las for other labs.
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