Construct optimization: We have engineered several new constructs for transformation in citrus genotypes. These include a ProBs314TBB-avrBs3: avrGf2 and a ProBs34TBB-avrBs3: avrGf2. In transient assays, these constructs elicited an earlier and stronger HR than constructs carrying avrGf1. We have also designed a construct which utilizes a portion of the promoter region of a citrus gene that demonstrates strong binding activity by PthA4 homologues. Transient assays demonstrate higher levels of activation than other constructs. Six new constructs are being used for stable transformation The constructs vary based upon the promoters used, the number of copies of the avrGf2 gene (single or multiple), the presence or absence of a terminator – nopaline synthase terminator (NOS T) upstream of the promoter and the plasmid used. Transformation summary In vitro germination experiments are ongoing with citrus varieties ‘Duncan’ grapefruit, ‘Ruby Red’ grapefruit and ‘Pineapple’ sweet orange for future epicotyl experiments. To date both epicotyl and cotyledon transformation experiments have been carried out with ‘Duncan’ grapefruit and ‘Pineapple’ sweet orange segments and the 6 new constructs designed with the avrGf2 gene. Sweet orange epicotyl and cotyledon transformation experiments have been carried out and a total of 1, 005 and 554 segments, respectively transformed with 5 of the 6 constructs. On the other hand 6 constructs have been used to transform altogether 3,623 grapefruit epicotyl segments and 5 constructs to transform 527 cotyledon segments in the transformation experiments. Shoots regenerated from transformed segments of sweet orange (53) and grapefruit (19) have been placed on rooting media. Putative sweet orange and grapefruit transgenic plants, 44 and 19 respectively originated from the regenerated shoots placed on rooting media have been placed in soil for acclimatization and will be tested via PCR for confirmation of integration of the transgene.
In the last few months, we continued to detect and identify the receptor and ligand. To ascertain the binding of CLas proteins to one immobilized ACP protein, the protein overlay assay was carried out on protein blots issued from 2D-SDS-PAGE. Non CLas-carrying insect total proteins were separated by 2D-SDS-PAGE. After 2D-SDS-PAGE, far-Western blot experiment was carried out. Comparisons between the stained electrophoretic profiles of ACP proteins in the gel and results of far-Western blot experiments on the membrane allowed the unambiguous election of protein spots from 2-DE gels for LC-MS/MS analysis. Some ACP proteins (receptor) were identified, which could play an important role in Clas adhesion to ACP cell. The function of these proteins was analyzed with bioinformatics. These genes were cloned, proteins were expressed, antibody against these proteins were made. Next, we expect to find the proteins of Clas (ligand) that adhesion to ACP cell using antibodies against identified ACP proteins, and certify the proteins interaction between ACP proteins (receptor) and Clas proteins (ligand). Overall, our research work is carried out according to project plan
The objectives of this project are: 1. Evaluate psyllid populations, HLB incidence and intensity, gene expression, tree growth, soil moisture, soil nutrients, foliar nutrients, and eventually yield in newly planted citrus blocks, 2. Assess separate contributions of vector control and foliar nutritional applications to the above parameters, 3. Evaluate the effectiveness of reflective mulch to (repel) ACP, 4. Provide economic analysis of costs and projected benefits and 5. Extend results to clientele. The experiment was planted 3-4 July on a 10-acre block on the A. Duda & Sons, Inc. farm in Hendry County south of LaBelle at 26.64315 degrees S. -81.45456 degrees W and 26 ft elevation. The experimental design of main plots is factorial RCB with 4 replicates and 4 treatments: insecticide alone, foliar nutrition alone, insecticide + nutrition, and untreated control. Each plot is split into two subplots, mulch and no mulch. Mulch is metalized (aluminized/reflective) polyethylene film shown in preliminary evaluations to repel Asian citrus psyllid and together with a drip irrigation/fertigation system increase citrus growth rate over the unmulched control. Flush inspection commenced and sticky cards were placed in the block on 13 Aug. Sticky cards are observed and replaced every other week for ACP and other common citrus pests. About 10 psyllids have been found on sticky card of which greater than 2/3 are in no mulch plots. In addition more ACP have been found on sticky cards in plots that do not have chemical control than plots that receive insecticides. Up to 10 young flush shoots are in inspected every 2 weeks on 15 trees in each of the 32 subplots. To date one ACP nymph has been found on flush. The first leaf samples were collected on October 17 for HLB testing of which one tested positive and is now undergoing a retest to insure the result is accurate. Monthly foliar nutrition applications were suspended after the early November spray in preparation for the winter dormant season. Leaf samples were collected November 7 for nutrient analysis. Normal grove care operations. These include one application of Intrepid for leaf miner control, one application of glyphosate for weed control in mulched plots, one application of glyphosate and Solicam in unmulched plots and Kocide once to control canker. All trees were desuckered and any low hanging limbs pruned to reduce the chance of herbicide damage. Some trees needed staking to prevent canopy break so new ties and stakes were added. Drip emitters were beginning to clog so all emitters were replaced in November with a clog resistant model as well as installing flush valves in December. Over the night of December 22-23 a freeze was experienced but the flood irrigation plan was executed so that no damage was incurred by the trees. New water sensor probes were tested and prepared for soil moisture data collection.
USDA-ARS-USHRL, Fort Pierce Florida has thus-far produced over 2,750 scion or rootstock plants transformed to express peptides that might mitigate HLB, and many additional plants are being produced. The more rapidly this germplasm can be evaluated, the sooner we will be able to identify transgenic strategies for controlling HLB. The purpose of this project is to support a high-throughput facility to evaluate transgenic citrus for HLB-resistance. Non-transgenic citrus can also be subjected to the screening program. CRDF funds are being used for the inoculation steps of the program. Briefly, individual plants are caged with infected psyllids for one week, and then housed for six months in a greenhouse with an open infestation of infected psyllids. Plants are then moved into a psyllid-free greenhouse and evaluated for growth, HLB-symptoms and Las titer. This report marks the end of the second quarter of the project, during which we have established the infrastructure for the screening program. A technician dedicated to the project has been hired, two small greenhouses for rearing psyllids have been completed and are functioning well, and 18 individually caged CLas-infected plants are being used to rear ACP for infestations. Psyllids will be available for challenging test plants in January. This screening program supports two USHRL projects funded by CRDF for transforming citrus.
The research team has continued to work with growers to collect data on yield, enhanced nutrient programs, and soil and leaf analyses. Initial analyses have been conducted. However, the current results are limited because only four growers have provided data thus far. As more data become available, the relationship between production and enhanced nutritional programs, if any, will become clearer.
The research team has continued to work with the growers to obtain information on the use of enhanced nutrient programs, soil and leaf analyses and yield. Two additional growers have provided data, and efforts to encourage participation from other growers continue. Data are being reformatted so that analyses can be conducted.
A talented postdoctoral fellow has been hired for the project and he has quickly begun making progress on research goals. Objective 1: Generate functional EFR variants (EFR+) recognizing both elf18-Xac and elf18-CLas. A. Mutagenesis of Arabidopsis EFR Conditions for optimal PCR random mutagenesis have been established and performed on the ectodomain of EFR. A library of approximately 1×106 clones, ready for transformation and screening, has been produced. A screening protocol has been established, whereby pools of 10 A. tumefaciens clones and infiltrated into N. benthamiana, and then scored for ROS induction in response to elf18-CLas. Screening has been initiated and we intend to screen approximately 3000 clones per week over the next three months. B. Natural variants of EFR Initial screening of a five species and cultivars of Brassicaceae has been performed, and we intend to obtain and screen a larger collection of species from several different genera for additional screening. Objective 2: Generate functional XA21-EFR chimera (XA21-EFRchim) recognizing axYS22-Xac. The PAMP receptors XA21 (from monocots) and EFR (from dicots) have been used to construct chimeric PAMP receptors. EFR-XA21 constructs have been produced to test the effectiveness of the XA21 cytoplasmic domain in signaling in dicots. This construct produces a ROS burst in response to elf18, to a similar degree as wild type EFR, when expressed transiently in N. benthamiana. Conversly, XA21-EFR and XA21 constructs have been produced and tested for responsiveness to ax21 in N. benthamiana. Thus far, a significant response has not been observed in the ROS burst assay; most likely due to a known issue of poor activity of the synthetic peptide (personal communication from Pam Ronald’s lab). Yet importantly, extracts from Xcv produced significant ROS burst with both XA21 and XA21-EFR constructs. Further evaluation of ax21 responsiveness using extracts from Xanthomonas euvesicatoria 85-10 (formerly, Xanthomonas campestris pv. vesicatoria) wild-type, or ax21 and raxST knockout strains (provided by Pam Ronald’s lab) that will enable us to conclude definitively on the functionality of XA21 and XA21-EFR is in progress. Transgenic Arabidopsis plants are being produced with XA21 or XA21-EFR to assess resistance to Xanthomonas campestris pv. campestris 8004 in dicots.
Our objective is to determine how Asian Citrus Psyllid (ACP) behavior is affected in response to infection of trees with the bacterium, Candidatis Liberibacter asiaticus (Las) that causes huanglongbing (HLB). We have been comparing ACP response to healthy (uninfected) versus diseased (infected) citrus. In previous experiments, we have determined that ACP adults initially settle on Las-infected plants as compared with uninfected plants. We hypothesized that while the Las-infected plants are initially attractive to ACP, after prolonged feeding, the ACP experience imbalanced nutrition and choose to seek a better host; therefore, moving to nearby uninfected plants. This may serve as a mechanism to spread the pathogen that causes HLB. In the previous quarter, we determined that ACP setting behavior may be affected by the age of the C. Las infection. Because of this, we used plants that were <5 month (young) or >12 months (old) following PCR confirmation of infection in settling choice assays. The choice scenarios we are testing include: control vs old infection, control vs, young infection, and young vs old infection. We have completed 4 replications of these settling experiments and are currently working on a fifth replication. Our results indicate the following: 1) When comparing response to the control vs old-infected plants, the ACP settle evenly between these treatments on the first day; however, at seven days ACP move to and preferentially colonize the infected plants. 2) When comparing the response to the control vs young-infected plants, the ACP settle on the young-infected plants and remain on this treatment for the duration of the 7-day experiment. 3) When comparing the response to young-infected vs old-infected plants, the ACP appear to settle evenly and stay evenly settled between these two treatments for the duration of the 7-day experiment. We will continue to conduct these settling choice tests until we have at least five replications. As an additional experiment, we will conduct similar settling bioassays with young-infected plants vs control plants in the presence of absence of large quantities of methyl salicylate (MeSA) to determine if exposure of ACP to large amounts of MeSA can mask the attractive odor of infected plants. The idea is to mask the main odorant we believe is responsible for attracting ACP to infected plants (MeSA) and thus develop a strategy to reduce pathogen spread. Also, we are in the beginning stages of developing a mathematical model to describe how the effect of infection of citrus plants, which subsequently affects behavior of ACP, may play a role in spread of the pathogen under field conditions.
In recent seasons, concurrent with freeze and drought episodes, symptomatic HLB-infected trees were much more affected by the extremes of temperature and moisture than trees without HLB. Symptoms exhibited by the stressed trees were excessive leaf loss and premature fruit drop even when HLB-infected trees were managed with enhanced nutritional programs which are thought to improve tree health of HLB-infected trees. This stress intolerance may be due to a loss of fibrous roots. To assess root status of HLB-affected trees, blocks of 2,307 three-yr-old Hamlin orange trees and 2,693 four-yr-old Valencia orange trees were surveyed visually and by real time PCR (PCR) to determine Las infection status. The incidence of Las-infected trees (pre-symptomatic PCR+, visually negative and symptomatic PCR+, visually positive) trees was 89% for the Hamlin block and 88% for the Valencia block. HLB+ trees had a 30 and 37% reduction in fibrous root mass density for pre-symptomatic and symptomatic trees, respectively, compared to HLB ‘ trees. In a second survey, 10- to 25-yr-old Valencia trees were identified within 3-6 months of canopy expression as HLB symptomatic (PCR+, visually positive) or non-symptomatic (PCR-, visually negative) in orchards located in the central ridge, south-central and southwest flatwoods. Pairs of HLB+ and HLB- trees were evaluated for PCR status, fibrous root mass density and Phytophthora nicotianae progagules in the rhizosphere soil. HLB+ trees had 27-40% lower fibrous root mass density and in one location higher P. nicotianae per root but Phytophthora populations per cm3 soil were high on both HLB+ and HLB- trees. Fibrous root loss results primarily from HLB damage which may be interacting with P. nicotianae.The root loss due to HLB is equal to or greater than 27% in our surveys of young and mature trees. In Brazil, Bassanezi and co-workers measured 973 mature trees of early, mid and late season sweet orange varieties and developed a model for crop loss in relation to visual canopy symptoms. Based on their model, trees in the early stages of HLB canopy decline are predicted to lose 30% of their crop, the same magnitude as the fibrous root loss we measured. An assessment of crop loss for groves in Florida treated with an enhanced nutritional program demonstrates similar magnitude of crop loss (23-39%) for HLB-affected Valencia trees. HLB-affected trees in these well managed Florida groves did not further decline in yield from 2009 to 2011 compared to the trees with HLB symptoms. We presume this is because additional loss of fibrous root density had not occurred since the initial onset of HLB symptoms 4 years prior.
Objective 1 (To define the role of chemotaxis in the location and early attachment to the leaf and fruit surface). Assays were performed to determine the ability of citrus bacterial canker (CBC) strains to respond to different stimuli. Cluster analysis showed an association between carbon source utilization and chemotaxis response. CBC strains were separated according to their host range. An in silico study of genes involved in chemotaxis was performed. Sequences for methyl-accepting chemotaxis proteins (MCPs) were conserved in all the xanthomonas strains evaluated. However, some MCPs were only present in a particular group of Xanthomonas strains. MCPs for the strains could be grouped according to host range demonstrating the connection with differential chemotaxis response. Chemotaxis response to different fractions from Chinese cabbage, Key lime and sweet orange leaves was compared. Chemotaxis response was greatest for apoplastic fluids, leaf extracts showed a moderate activity, and leaf washes showed no activity. Apoplast fluid from sweet orange most stimulated movement in wide host range canker strain 306, cabbage fluid did the same for X. campestris. Key lime apoplast fluid most affected X. alfalfae subsp. citrumelonis. Narrow host range strains of CBC (Aw and A*) showed little response to any of the leaf fractions from any of the plant species. Objective 2 (To investigate bifofilm formation and composition and its relationship with bacterial motility structures in different CBC strains and comparison to non canker causing xanthomonads). Protein analysis showed no qualitative differences in bacteria appendages among the Xanthomonas strains. Alterations in gene expression of type IV pilus, and flagellum components are under evaluation with qPCR primers for assay of bacterial colonies at different stages of motility swarming, planktonic or biofilm-forming.
The overall goal of this project was to transfer disease resistance technology from Arabidopsis to citrus. Two specific aims were proposed in the original proposal. One was to overexpress the Arabidopsis MAP kinase kinase 7 (MKK7) gene in citrus to increase disease resistance (Transgenic approach), and the other was to select for citrus mutants with increased disease resistance (Non-transgenic approach). For specific aim #1, we have generated not only transgenic citrus plants overexpressing MKK7 but also transgenic plants overexpressing several other Arabidopsis disease resistance genes including NPR1, NAC1, MOD1, and EDS5. While disease resistance test for most of the transgenic plants is underway, transgenic plants overexpressing NPR1 were found to have increased resistance to citrus canker (see below). For specific aim #2, we have tested different citrus plant materials for mutagenesis, including calli, hypocotyls, and seeds. Chemical genetic screens have been carried out using these materials. In the last year of the project, we started a direct genetic screen for citrus greening-resistant varieties using grapefruit seeds mutated with gamma ray irradiation. This screen is still ongoing. During the project, we not only tried to accomplish the originally proposed work, but also explored the recently discovered disease resistance technology in the model plant Arabidopsis. At the end of the project, several significant results have been obtained. (1) We found that overexpression of the Arabidopsis NPR1 gene, which is a key regulator of systemic acquired resistance (SAR), in citrus increases resistance to citrus canker. This result has been published in European Journal of Plant Pathology. Furthermore, we found that the transgenic plants overexpressing NPR1 did not have increased resistance to citrus greening. (2) We found that the citrus canker-causing bacterial pathogen Xanthomonas citri subsp. citri (Xcc) is a nonhost pathogen of the model plant Arabidopsis. We discovered that Xcc neither grows nor declines in Arabidopsis, but induces strong defense gene expression. This result has been published in PLoS ONE. (3) Using the Arabidopsis-Xcc pathosystem, we found that the salicylic acid (SA) signaling pathway contributes to nonhost resistance against Xcc in Arabidopsis. Several genes of the SA signaling pathway were found to contribute to nonhost resistance against Xcc. (4) We found a group of novel genes, which play critical roles in nonhost resistance against Xcc in Arabidopsis. We revealed that Xcc grows significantly more in mutants of these genes. For instance, in one of these mutants, Xcc grows about 50-fold more than in the wild type, suggesting that the corresponding gene is a critical regulator of nonhost resistance against Xcc. More importantly, we found that overexpression of this gene confers resistance to several virulent bacterial pathogens; therefore, the newly discovered nonhost resistance genes hold great potential for generating disease-resistant citrus varieties. (5) We found that exogenous NAD+, which induces strong SAR in Arabidopsis, activates strong resistance to citrus canker, suggesting that the NAD+-mediated defense signaling pathway is highly effective against citrus diseases. Therefore, components we have identified in the NAD+-mediated signaling pathway could be used to engineer resistance to citrus greening and/or canker.
This is a 4-year project with 2 main objectives: (1) Over-express the Arabidopsis MAP kinase kinase 7 (AtMKK7) gene in citrus to increase disease resistance (Transgenic approach). (2) Select for citrus mutants with increased disease resistance (Non-transgenic approach). For objective 1, the transgenic citrus plants overexpressing the Arabidopsis MKK7 (AtMKK7) gene are under disease resistance test for citrus canker and greening. While waiting for the resistance test result, we expanded the project to identify genes that confer nonhost resistance to the citrus canker causing bacterial pathogen Xanthomonas citri subsp. citri (Xcc). We have previously established an Arabidopsis-Xcc pathosystem with the support of a USDA special grant, and have found that mutants of the SA signaling pathway are more susceptible to Xcc. These results have been published in PLoS ONE. Using the Arabidopsis-Xcc pathosystem, we screened available Arabidopsis mutants and identified a group of novel genes conferring nonhost resistance against citrus canker. Importantly, we found that overexpression of one of these nonhost resistance genes increases resistance to several virulent bacterial pathogens. Furthermore, we have generated citrus transgenic plants that express two salicylic acid (SA) biosynthesis genes. These transgenic plants are expected to accumulate more SA, which should transfer to stronger resistance to citrus canker and/or greening. We are testing the SA levels in the transgenic plants. For objective 2, we are continuing the direct genetic screen for citrus greening-resistant varieties. Gamma ray-irradiated Ray Ruby grapefruit seeds were germinated in soil and the resulting seedlings were inoculated with psyllids carrying greening bacteria. While adding more seedlings from gamma ray-irradiated Ray Ruby grapefruit seeds into the screen, seedlings developing greening symptoms were removed from the screen. We are watching the development of greening symptoms on the remaining seedlings.
This report covers the period of the last three months of 2012. Citrus Core Transformation Facility continued to operate at the high level and produced transgenic Citrus plants for multiple orders. The work continued toward a completion of ‘Y’ order and following transgenic Carrizo plants were produced: two plants with the gene from Y141 plasmid; 27 plants with the gene from the Y109 plasmid; and two plants with the gene from Y150 plasmid. Two Duncan plants were produced with the AZI1 gene. Two Duncan plants were produced with the gene from SF1 vector. Three Duncan plants were produced with the DPR1 gene. Continued experiments on some old orders yielded one Duncan plant with the EDS5 gene, four Duncan plants with the gene from WG20-7 vector, and five Duncan plants with the gene from WG19-5 vector. Eleven Duncan plants were produced transformed with genes from MOG800 vector. Seven Duncan plants were transformed with the AtBI gene. One Duncan plant with the CIV2 gene was also produced. The work on newer orders resulted in production of transgenic Duncan plants carrying genes from different vectors: four from the X4, ten from the X7, two from the X11, one from the X16, five from the X19, and five from the X20. The CCTF received six more new orders to produce transgenic plants carrying genes from vectors named pN4, pN5, pN7, pN9, pN12, and pN18. All of these orders requested production of transgenic Duncan plants. Cultures of Agrobacterium cells carrying these six binary vectors were already produced and are ready to be used in co-incubation experiments. With the plenty of recent and the newest orders, the facility will continue to operate at full capacity also working on full completion of older orders.
The overall objective of this project is to develop and use a high-throughput system to screen for chemicals that disrupt interactions in a model of the ACP/HLB/Citrus system that uses the related bacterium Candidatus Liberibacter psyllaurous (CLps) which causes psyllid yellows of tomato. Previous work focused on development of a system for the model plant Arabidopsis thaliana which has the best developed genetics of any plant and has been used in previous chemical genomics experiments. However, repeated attempts to infect Arabidopsis plants grown in solid culture media, liquid culture media, or hydroponics were not successful. Only plants grown in soil were infected by psyllid nymphs. The percentage of Arabidopsis plants infected after inoculation by 10 psyllids was as low as 15% in some experiments making this a difficult system for chemical genomics. It is of great interest that wild type Arabidopsis plants infected by CLps show no symptoms of infection – they appear tolerant despite a relatively high bacterial titer as measured by qPCR. During the current quarter, we investigated the response to CLps infection of Arabidopsis lines homozygous for mutations in various pathogen defense response genes. We evaluated 5 mutant and Col-0 and Ler-1 wild type Arabidopsis lines responses to CLps infection. Out of 18 plants of each line, 15 plants were infested with CLps positive psyllid-nymphs and 3 were non-infested control plants. All plants were grown under short day conditions to delay flowering. From each plant, tissues were collected at different time intervals in the following manner: 2 leaves at 2.5weeks post infestation and 1 leaf, 3 stem segments and the inflorescence at 4-5weeks post infestation (wpi). Results for harvested tissues at 2.5 weeks indicated that some plants of each line were CLps positive but appeared normal in phenotype. Two mutant lines had highest number of CLps positive plants (5 of 15). Interestingly, at 5wpi CLPs-positive plants of some mutant lines showed a strong phenotype including leaf discoloration, stunted leaf growth, delayed bolting, and short internodes. For one mutant line, at 5 wpi 12 of 15 plants were found positive for CLps with average Ct of 25.22 for stems and Ct of 26.47 for leaves. In this line, all 12 CLps positive plants showed the distinct phenotype whereas 3 infested but CLps negative plants had a normal phenotype similar to that of the non-infested plants. The number of plants of each line tested so far is small, but the results appear clear cut. To correlate these symptoms with CLps infection, and confirm these results a much larger experiment has been initiated. In this experiment we are testing one mutant line (that with highest number of CLps positive plants) in response to three treatments, non-infested, infested with CLps positive psyllids and infested with CLps negative psyllids. If confirmed, this result implies that the apparent tolerance of Arabidopsis to CLps depends upon expression of specific defense responses. It may be important to determine the proportion of living vs dead bacteria in various Arabidopsis lines. The larger experiment initiated in December will be used to compare gene expression of normal vs mutant lines following feeding by CLps positive and negative psyllids.
The overall objective of this project is to develop and use a high-throughput system to screen for chemicals that disrupt interactions in a model of the ACP/HLB/Citrus system that uses the related bacterium Candidatus Liberibacter psyllaurous (CLps) which causes psyllid yellows of tomato. Previous work was focused on development of a system for the model plant Arabidopsis thaliana which has the best developed genetics of any plant and has been used in previous chemical genomics experiments. However, repeated attempts to infect Arabidopsis plants grown in solid culture media, liquid culture media, or hydroponics were not successful. Only plants grown in soil were infected by psyllid nymphs. The percentage of of Arabidopsis plants infected after inoculation by 10 psyllids was as low as 15% in some experiments making this a difficult system for chemical genomics. We continued to work on developing a chemical genomics system using tomato petiole-leaf tissues. We also investigated use of a transgenic tomato carrying a marker gene (GUS) driven by a pathogen responsive promoter (CaBP22) in this system. A similar system developed in Arabidopsis has facilitated identification of defense pathway activating chemical compounds. In earlier experiments we found that Tomato (Moneymaker) detached leaves infested with CLps positive psyllids typically have a high percentage of CLps positive leaves which provides an advantage to test large number of chemicals for their effect on CLps in a high-throughput manner. Based on this experiment, we investigated the chemical uptake in detached leaves of CaBP22 promoter ‘ GUS transgenic cherry tomato to confirm if chemicals can be absorbed through detached petioles. Within 24-48hrs of chemical incubation, detached leaves incubated in 500uM of chemical showed yellow patches on leaflets indicating phytotoxic effect. In Arabidopsis, high concentrations of the tested chemical also have phytotoxic effects. Detached leaves exposed to 100uM or water and DMSO controls did not show any changes. Two segments of petiole were collected from each detached tomato leaf, a bottom segment of petiole placed in chemical/ water (and so not directly exposed to psyllids) was tested for CLps infection and the petiole segment exposed to psyllids was collected for testing GUS expression. We will test these segments for CLps infection and confirm the chemical uptake using the GUS reporter gene. If chemical uptake is confirmed, then the tomato leaf system is suitable for chemical genomics experiments. During this quarterly report period we mainly focused on a related (but separately funded) project that would facilitate testing chemicals on citrus. Sweet orange seedlings were exposed to experimental chemicals that induce plant defense responses in other Arabidopsis and some other plants. Tissue samples were collected for qPCR experiments to measure changes in expression of specific defense response genes. RNA was isolated from collected samples. Gene expression patterns of Arabidopsis in response to these chemicals were studied and Citrus orthologs of Arabidopsis genes showing higher fold changes were identified from genome sequence databases and primers obtained. qPCR to evaluate citrus gene expression has not been completed.
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