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.
Obj 1: As a first step to validate bioinformatically predicted effectors identified from mining of our PAVE database, we selected 20 interesting candidates that were detected in both the ACP and PoP transcriptomes. Primers were designed around the most conserved nucleotides in each sequence and RT-PCR was performed using PoP cDNA. To date, 12 of those transcripts are confirmed to be expressed. Confirmation of the remaining 8, as well as all genes in ACP by RT-PCR is ongoing. Obj 2: To initiate yeast-2 hybrid studies to elucidate protein-protein interactions important in HLB disease, 500 guts from Las-infected ACP, and 500 guts and 1000 salivary glands from uninfected ACP have been isolated. Good quality RNA has been isolated from all of these organs and cDNA library preparation is underway. Obj 3: For proteomic identification of putative effectors proteins from 50 guts and 250 salivary glands of infected and noninfected potato psyllid adults were separated by SDS-PAGE (10’14% acrylamide). After trypsin digestion, peptides were analyzed by nanoLC-LTQ-Orbitrap (Thermoelectron) mass spectrometry. We identified 828 and 358 proteins in the gut using all the PoP and ACP translated PAVE databases, respectively. Using a 2 fold change cut-off, 45% of the proteins identified using the PoP transcripts (373/828) and 40% of proteins using the ACP transcripts (142/358) showed differential abundance in the uninfected proteome compared to the Lso-infected proteome. We identified 729 and 357 proteins in the salivary gland using all the PoP and ACP translated PAVE databases, respectively. Using a 2 fold change cut-off, 58% of the proteins identified using the PoP transcripts (425/729) and 55% of proteins using the ACP transcripts (198/357) showed differential abundance in the uninfected proteome compared to the Lso-infected proteome. Obj 4: Using data from objectives 1 and 3 (above), we have selected two psyllid and two Liberibacter genes for functional characterization by RNAi analysis using the oral delivery method. Good quality dsRNA has been made using the MEGAscript RNAi kit and feeding studies are underway. In addition to qPCR validation of mRNA knockdown using the psyllid COI gene for normalization, we have designed novel bioassays to confirm predicted functional roles in the transmission process. Psyllids can survive up to 2 weeks in buffer only controls. Selection of salivary gland-specific genes for RNAi analysis by microinjection is underway. We have optimized the injection procedure with a novel pre-puncturing system using a fine tungsten wire followed by insertion of fluid-filled needle. Currently, psyllid survivability on buffer only controls is 50% and we hope to improve upon this with by exploring modifications.
In this project, we proposed three aims in order to identify, characterize, and make use of citrus genes with a potential role in SA-mediated defense in engineering resistance to canker and greening diseases in citrus plants. Among the three proposed aims, the first aim has been completed, the second aim is about to be finished, and the third aim needs longer time to complete due to long-term growth nature of citrus plants. So far we have identified at least one citrus SA gene that could have effects on canker disease when overexpressed. Additional citrus transgenic plants are under further production and defense tests. We believe that we have met the expectations of the project and here provide a summary of the project. Objective 1: Identify genes positively regulating SA-mediated defense in citrus We identified over 10 citrus SA homologues via bioinformatics analysis. We used an RT-PCR approach to clone 10 full-length cDNA for the citrus SA homologues, which were further cloned into a binary vector pBIN19ARSplus for making transgenic plants in Arabidopsis and citrus. We have also finished collecting citrus tissues infected with Ca. L. asiaticus in a time course. qRT-PCR analysis with these samples was conducted for some SA genes. Our results showed that expression of at least one of the genes, ctNDR1, showed an induction upon HLB infection, suggesting a possible role of ctNDR1 in defense against HLB. Objectives 2: Complement Arabidopsis SA mutants with corresponding citrus homologues All 10 SA citrus genes were used to transform Arabidopsis plants, either complementing the corresponding mutants or overexpressing in wild type. We obtained T0 seeds for these constructs and selected most T0 seeds for T1 transgenic plants. Some seeds were further selected for homozygotes at the T2 generation. Most of the transgenic plants were tested for disease resistance to the infection of Pseudomonas syringae. So far, we found that at least two of the constructs ctNDR1 and ctEDS5 showed some level of disease resistance. However, there was no significantly increased resistance in CtNPR1 transformed Col or npr1-1 mutant and CtPAD4 transformed Col or pad4-1 mutant. Additional tests are undergoing for other transgenic plants. We have done more detailed characterization of ctNDR1 plants, which was summarized in a previous progress report (April 2012). A manuscript for this work should soon be submitted for a consideration of publication. Objectives 3: Assess the roles of SA regulators in controlling disease resistance in citrus We have so far produced transgenic plants for ctNPR1, ctEDS5, ctPAD4, and ctNDR1 and the presence of the transgenes in these plants were confirmed by PCR. In addition, we have tested disease resistance of ctNDR1 plants with Xanthomonas citri subsp (Xac), the causal agent for citrus canker disease, and found that overexpressing this gene confers some level of resistance to the strain. We will further test if ctNDR confers resistance to greening disease. In addition, we will continue to produce transgenic plants overexpressing other SA genes and selected transgenic plants will be tested for resistance to canker and greening diseases. These activities will be conducted after the end of the grant period.
In this project, we proposed three aims in order to identify, characterize, and make use of citrus genes with a potential role in SA-mediated defense in engineering resistance to canker and greening diseases in citrus plants. Among the three proposed aims, the first aim has been completed, the second aim is about to be finished, and the third aim needs longer time to complete due to long-term growth nature of citrus plants. So far we have identified at least one citrus SA gene that could have effects on canker disease when overexpressed. Additional citrus transgenic plants are under further production and defense tests. We believe that we have met the expectations of the project and here provide a summary of the project. Objective 1: Identify genes positively regulating SA-mediated defense in citrus We identified over 10 citrus SA homologues via bioinformatics analysis. We used an RT-PCR approach to clone 10 full-length cDNA for the citrus SA homologues, which were further cloned into a binary vector pBIN19ARSplus for making transgenic plants in Arabidopsis and citrus. We have also finished collecting citrus tissues infected with Ca. L. asiaticus in a time course. qRT-PCR analysis with these samples was conducted for some SA genes. Our results showed that expression of at least one of the genes, ctNDR1, showed an induction upon HLB infection, suggesting a possible role of ctNDR1 in defense against HLB. Objectives 2: Complement Arabidopsis SA mutants with corresponding citrus homologues All 10 SA citrus genes were used to transform Arabidopsis plants, either complementing the corresponding mutants or overexpressing in wild type. We obtained T0 seeds for these constructs and selected most T0 seeds for T1 transgenic plants. Some seeds were further selected for homozygotes at the T2 generation. Most of the transgenic plants were tested for disease resistance to the infection of Pseudomonas syringae. So far, we found that at least two of the constructs ctNDR1 and ctEDS5 showed some level of disease resistance. However, there was no significantly increased resistance in CtNPR1 transformed Col or npr1-1 mutant and CtPAD4 transformed Col or pad4-1 mutant. Additional tests are undergoing for other transgenic plants. We have done more detailed characterization of ctNDR1 plants, which was summarized in a previous progress report (April 2012). A manuscript for this work should soon be submitted for a consideration of publication. Objectives 3: Assess the roles of SA regulators in controlling disease resistance in citrus We have so far produced transgenic plants for ctNPR1, ctEDS5, ctPAD4, and ctNDR1 and the presence of the transgenes in these plants were confirmed by PCR. In addition, we have tested disease resistance of ctNDR1 plants with Xanthomonas citri subsp (Xac), the causal agent for citrus canker disease, and found that overexpressing this gene confers some level of resistance to the strain. We will further test if ctNDR confers resistance to greening disease. In addition, we will continue to produce transgenic plants overexpressing other SA genes and selected transgenic plants will be tested for resistance to canker and greening diseases. These activities will be conducted after the end of the grant period.
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). First group of samples for RNA-Seq were selected at Picos Farm at Fort Pierce, including three Jackson grapefruit plants (resistant/tolerant) and three Marsh grapefruit plants (susceptible). Total RNA has been extracted from the new flush leaf samples of each of these six citrus plants. The qualified RNAs are being used to construct the library for Illumina sequencing. We have obtained the sequences from five of the six samples; a total of ca. 269 millions reads and ca. 49 GB nucleotide sequences were just obtained, and we are going to conduct intensive bioinformatic analysis from now on. While waiting for the sequences, we conducted a genome wide prediction of citrus resistance genes using genome annotated genes and unigenes of Citrus clementine (Cc) and C. sinensis (Cs) from Citrus Genome Database. We identified 607, 484 and 499 genes containing the NBS domain (NB-ARC, PF00931) in C. clementine, C. sinensis and Csc respectively using pfam_scan. There were 426, 250 and 322 genes after filtering with NBS domain coverage over 80% (230 bp over 287 bp of NB-ARC domain). There are more NBS-containing proteins in the genome of C. clementine than that of C. sinensis. Some of the NBS genes were found to have an expression. 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 EST varied from 1 to 25. We predicted the NBS orthologs of citrus with inparanoid and multiparanoid. A total of 131 orthologs were identified, which contains 213, 146 and 191 NBS genes for C. clementine, C. sinensis and Csc, respectively. A total of 241 NBS genes were specific to C. clementine. Although the total number of NBS genes were similar in the two varieties of sweet orange, there were 226 and 198 variety-specific NBS genes assigned to each, respectively. A total of 318 out of 499 NBS genes in sweet orange could be mapped to the 9 chromosomes. The NBS genes clustering distributed in chromosome 1, 3 and 5 in sweet orange. There are still 181 NBS genes that could not be mapped to the known chromosomes.
The goal of this project is to develop a transcriptomic toolbox to elucidate psyllid-Ca. Liberibacter (Las and Lso) interactions and associated effectors that can be used for management of citrus greening disease. In this project we constructed 14 Illumina paired-end libraries including uninfected adults, infected adults, uninfected nymphs, infected nymphs, uninfected guts, infected guts, uninfected salivary glands and infected salivary glands generating 72,539 and 81,682 unique transcripts (unitrans) from Asian citrus psyllid (ACP) and potato psyllid (PP), respectively. Secondly, we developed a user-friendly database containing the assemblies ‘DcWN’ and ‘BcWN’ from the eight libraries yielding a total of 45,799 and 81,682 unitrans. Of the 45,799 total unitrans, 17,988 unitrans (39%) were thus annotated, which comprised 35.6 Mb of sequence with an average unitrans length of 1,981 bp, ranging from 150 bp to 26,540 bp. Of the 81,682 total unitrans, 16,462 unitrans (20%) were annotated. Unitrans resources comprise 28.9 Mb of sequence with a N50 length of 1,390 bp and an average unitrans length of 1,756 bp, ranging from 100 bp to 27,405 bp. Comparison of expression data between the infected and uninfected Asian citrus psyllid highlights several differences. We have also been able to identify genes involved in RNAi based silencing. Results from comparative transcriptomic analyses showed that ACP and PP are more similar than dissimilar, and suggesting that researchers in quarantined areas without access to ACP can use the PP as a surrogate model organism to advance research efforts. To our knowledge this is the first report elucidating a detailed transcriptome analysis of ACP and PP. The data provides insights about vector-pathogen relationships at the molecular genomic and gene expression levels. This effort provides an invaluable resource to aid in effector gene identification relevant to establishment and transmission pathways in the psyllid host. Using the databases, putative gene families common to both psyllids and also other insect genomes were identified, as well as a suite of unique sequences that when taken together with the Ca. Liberibacter genome sequences can make possible predictions about ‘interactors’. In addition a pattern of responses to Ca. Liberibacter infection indicated that that Ca. Liberibacter infection negatively affects psyllid nymphs to a higher degree than adults. Additionally, transcriptomic data show it can be used to study obligate endosymbionts based on presence of transcripts sharing high nucleotide sequence similarity to Wolbachia sp. and Carsonella. In silico gene expression analyses identified several psyllid and bacterial genes putatively involved in psyllid development, Ca. Liberibacter transmission and pathogenesis (adhesion, nutrition, pathogenicity, lytic function, viral functions). Based on differential expression patterns of specific gene ontology functions such as iron metabolism and immune responses, we identified the candidates transferrin and Caspar, that could be investigated further to understand their predicted roles in transmission and bacterial survival. Also we identified differentially expressed transposon-like transcripts in infected and uninfected libraries. Several transposable element-related transcripts are differentially expressed in the Ca. Liberibacter infected libraries that may be significant by aiding Ca. Liberibacter a competitive advantage over the psyllid host. Experiments to validate candidate ‘interactors’ and test putative transcript functionality are underway using various strategies including transcript knockouts. We have optimized feeding assays for dsRNA delivery, and qPCR analysis of gene expression of gut genes, and are optimizing microinjection as the next step for monitoring knockouts directed at salivary gland/oral box-secretome effectors.
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