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.
MtCOI haplotyping. Additional samples are gradually received for mtCOI haplotyping. A total of 250 samples were analyzed, including from a center of diversity in Asia, and elsewhere are to explore baseline diversities within and between different locations, in an attempt to relate populations from the US and elsewhere in the Western Hemisphere to those from a primary region of endemism. Sequences (~1300 bases) are remarkably similar, suggesting the need for a different marker. This result was surprising, considering the widespread use of the COI for homopteran bar-coding. Time-course and transmission studies were established to assess transmission frequency in relation to qPCR and/or dot blot hybridization detection of Ca. Liberibacter solanacearum (AZ) and in Ca. L. asiaticus (FL) in individual psyllids reared on infected plant material. Single potato psyllids reared on infected tomato and given a range of inoculation access feeds on tomato from 30 min, and 1,2, 4hr-transmitted Ca. L. solanacearum 20, 35, 30, and 75% of the time (20 plants per rep). When five psyllids were used transmission frequency was 5, 35, 25, and 70%, respectively. Studies suggest that once acquired psyllids transmit at relatively high frequency (70%); tests are underway for potato psyllid at 8,12,and 24 hrs. The goal is to select individuals for light microscopy, and SEM-TEM ultrastructural observations to understand the dynamic relationship over a range of different AAP and IAPs. IAP studies are underway for Ca. Lsol using tomato seedlings for bioassay, together with PCR or qPCR monitoring, all difficult undertakings owing to the apparent uneven distribution of bacteria in plants, which confounds reliable detection for localization studies. Transmission and scanning electron microscope studies focused on large monocultures [interpreted as thick biofilms] of rod-shaped, fastidious bacteria that are consistently associated with the alimentary canal, from the oral box (including salivary glands) to the posterior midgut, of infected but not uninfected psyllids, and most consistently observed in 3rd instar and older psyllids. In addition, studies have revealed the dynamic behavior in the positions that the intestine assumes within the abdomen of live psyllids. As with the causal, bacterial agent for Pierce’s disease, attempts to reliably implement FISH for determining the time-course proliferation and gross anatomical affinities of the target pathogen within the psyllid vector body remain tentative. We are implementing colloidal gold – DNA hybridization for ultrastructural studies. 100nm thick, plastic Z-section libraries of the oral box have been made from infected and non-infected potato psyllids to map the tissue/organ organization where bacteria reside. The net accomplishments are visualization of Ca. Liberibacter in the newly resolved anatomical structures, and constructing models for mode and pathway of transmission. In research designed to prove Koch`s postulates for CLso, we determined that infected plant symptoms do not correlate with signal detection in qPCR. We have developed a culture technique that sustains the bacterium for a period of two weeks in liquid medium. We were able to show that clean Psyllids that were never exposed to CLso when fed on artificial feeding solution containing infected psyllid gut extracts are able to acquire CLso detectable by regular PCR and qPCR for OMP gene of CLso. Using TEM, we were able to determine average length of this bacterium to be 3.52um and average diameter to be 0.15um. qPCR on near pure cultures show bacteria survive and reproduce. Unusual and polymorphic shapes of CLso cells observed in and on internal psyllid organs suggest that they do not form a typical septum as is common during binary fission of many bacteria. Two-three manuscripts reporting these results will be submitted.
In this continuation we are (i) utilizing mined EST data in PAVE to validate bioinformatically predicted effectors using RNAi knock down analyses (feeding, microinjection), together with FISH localization of mRNA, and qPCR quantification; additional putative protein effectors will be identified in the secretome (proteome of dissected salivary glands and guts; candidate EST-expressed proteins), (ii) initiating efforts to implement yeast-2 hybrid to elucidate protein-protein interactors in ACP adults and Las expression libraries (psyllids courtesy K. Stelinski), and (iii) validation by in vivo pull down assays. Classes of targets of interest will be based on comparative SEM-TEM evidence together with transcriptomic- and bioinformatics-driven (EST) predictions thus far include the psyllid secretome, vector innate immunity and RNA silencing pathways, nutrition-metabolism, and Las adhesion and virulence-pathogenicity factors predicted based on the Las genome sequence. Purative effector proteins will thereby be identified using a multi-pronged, progressive approach. The goal is to implement new knowledge of the key effectors identified here to inform genetics based approaches for abating vital secretome functions required for psyllid livelihood and nourishment, (ii) Las infection and circulation, and (ii) transmission to control citrus greening disease. Comparative analyses of ACP and PP psyllid transcriptomes revealed that ~60% of the transcripts were common to both species suggestive of roles in core growth and developmental processes which are ideal targets for RNA interference (RNAi) with the goal to interfere with psyllid survivability. These kinds of target are also good for initial optimization of RNAi using mortality bioassays. A number of these genes were selected and experimentally validated by RT-PCR in ACP adults. Additionally, in silico expressional analyses of ACP and PP transcripts in response to Liberibacter infection have been conducted, resulting in the identification of a large number of up- and down-regulated transcripts (>2 fold) in infected psyllid nymphs and adults. BLAST (NCBI) searches and gene ontology gene predictions indicate these genes are likely involved in key psyllid-Liberibacter interactions such as in virulence, adhesion, and immunity, which could be lucrative targets for RNAi to interfere with survival, propagation, circulation, pathogenicity, and ultimately transmission of Ca. Liberibacter. RNAi assays are under development employing a system developed previously for whitefly artificial feeding to orally deliver dsRNA corresponding to gut targets. The dsRNAs for selected targets are synthesized using in vitro transcription and the MEGAscript RNAi kit. Currently adult psyllids survive up to 5 days in this system. We also developed a quantitative qPCR detection method using the psyllid COI gene for normalization. Information about the molecular basis of key psyllid-Liberibacter interactions is lacking with respect to entry, metabolic pathways use, and immune system effectors and interactions with bacterial proteins. Proteins from guts and salivary glands of infected and noninfected potato psyllid adults were separated by SDS-PAGE (10’14% acrylamide). Each gel lane was digested with trypsin and peptides were analyzed by nanoLC-LTQ-Orbitrap (Thermoelectron) mass spectrometry. We identified 795 proteins in the gut. Of those, 212 proteins showed >1.5 fold increase in expression in infected compared to noninfected guts. Among these are a number of putative effectors with direct implication in bacterial transport and movement in the circulative, propagative pathway that have been selected for mRNA validation and qPCR analysis of expression levels in infected and uninfected adult ACP and PoP psyllids.
Expression in citrus of dsRNA targeting a psyllid gene, through use of the paratransgenic CTV expression vector, was further characterized. Our analysis showed that mortality of psyllids feeding on citrus producing target dsRNAs was directly correlated with accumulation of total psyllid gene RNA (ssRNA + dsRNA) produced within the leaf tissue. As much as 80 to 90% mortality of adult psyllids was observed after 6 days of feeding on leaves with the highest level of psyllid target gene RNA. Citrus leaves expressing RNA from the green fluorescent protein (GFP) cloned into the CTV expression vector induced no mortality in adult psyllids. These results support the hypothesis that mortality is associated with psyllid gene specific dsRNA ingestion. Currently experiments are being conducted on performance of all psyllid life cycle stages.
Progress with the rapid flowering system (pvc pipe scaffolding system) in the greenhouse: Selected transgenic plants produced from juvenile explant, budded to precocious tetraploid rootstocks in airpots are growing well in our RES system, with some plants reaching 8 feet in height. Additional transgenics were propagated onto additional new rootstocks expected to reduce juvenility, including the somatic hybrid Amblycarpa + Flying Dragon. The goal is to reduce juvenility by several years to accelerate flowering and fruiting of the transgenic plants. Experiments to efficiently stack promising transgenes are underway. The first transformation experiments using the two-transgene Gateway based cloned construct combining our best transgene for HLB resistance (NPR-1 from Arabidopsis) with our best transgene against canker that also has some affect on HLB (the synthetic CEME lytic peptide gene) were initiated, and so far 30 putative transgenic lines of the sweet orange cultivars Hamlin and Valencia have been regenerated. These plantlets have been micrografted to Carrizo rootstock. The goal is to provide stable resistance to both HLB and canker, with transgene backup to prevent Liberibacter from overcoming single transgene resistance. Correlating transgene expression with disease resistance response: We continued work to optimize an ELISA protocol to detect lytic peptide in transgenic Citrus plants using the LIMA antibody. This protocol has should be useful for evaluating transgenic plants containing either LIMA or CEME antimicrobials, using the same antibody. Since most of our constructs have the C-myc tag, ELISA and Western blot protocols have been optimized for large scale rapid screening of the transgenic plants to identify those with maximum transgene expression. Improved transformation methodology (for seedless or recalcitrant cultivars, and eventually marker-free consumer-friendly transformation): A vector containing a dual T-DNA border has been constructed. To test the vector functionality and determine T-DNA segregation, we have incorporated a visual Anthocyanin expressing gene from Grape (VVMYB) into one of the T-DNA. This gene on expression turns cells purple. The other T-DNA contains a fusion negative-positive selectable marker gene for selection (codA/nptII; Vector 1). We are currently constructing another fusion negative-positive selectable marker gene, by replacing the nptII gene with a gene that encodes for resistance to the antibiotic hygromycin (hptII). This construct will be used for transformation of citrus cell suspension cultures (Vector 2).
Dr. Cecilia Zapata, PI, resigned August 2012. Before leaving, we planned for reduced effort until a replacement could be found. Dr. Vladimir Orbovic agreed to assist with oversight of the mature tissue transformation facility in the interim. Below is the quarterly report Dr. Orbovic submitted. In the first three months of the funding period, Mature Tissue Transformation Laboratory (MTTL) has undergone big changes. The person who supervised the Lab for the last three years has left that post in the beginning of September. In the anticipation of prolonged period without managerial supervision for MTTL, departing supervisor made a decision to discard high percentage of plants from the growth room to prevent accumulation of unused plants. The transformation experiments were scheduled at the rate of one per month. However, temporary supervisor revised the plan up to two experiments per month. To accommodate such change, certain batches of plants that were used a source of explants only once were not discarded as planned. Also, some of the smaller rootstock plants left for practice and as surplus were transplanted and will serve as an additional batch of rootstock plants. Throughout this period, nine co-incubation experiments were performed. Four of those experiments were done with Hamlin explants, four with Valencia explants, and one with Pineapple sweet orange explants. In the Hamlin experiments, 2390 explants were cut for treatment with Agrobacterium; 2520 explants were used in Valencia experiments, and 690 explants were used in Pineapple experiment. Here are the results of GUS assays: 270 shoots harvested from different experiments with Hamlin were tested and two were positive. Out of 210 tested shoots of Valencia harvested from different experiments, five were positive. And finally, out of 68 shoots of Pineapple orange harvested from two experiments, two were positive. One of two positive Valencia shoots died upon grafting. Other positive shoots appear healthy and will be moved to growth room soon. These results mark a milestone as all three commercially important cultivars of sweet orange were successfully transformed. There are four Ray Ruby plants completely cleaned from microorganisms and ready to become source of shoots for production of branches. These Ray Ruby plants were obtained from USDA as ‘clean’ although additional testing in MTTL has shown that they did harbor some microorganisms. Repeated micro-grafting of meristem regions to new and clean rootstock plants resulted in selection of plants that were purged of any pests.
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