The transgenic plants to be developed for this project are now growing in two different locations in secure greenhouses and growth chambers. Seven independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct were shipped from the Citrus Transformation Facility at the University of Florida Citrus Research and Education Center at Lake Alfred, FL, to Dr. McNellis’ lab at the Pennsylvania State University at University Park, PA, in early October, 2014. An additional eight independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct were shipped to Dr. Tim Gottwald’s lab at the United States Horticultural Laboratory in Fort Pierce, Florida. The plants at both locations are growing well. In summary, a total of 15 independent transgenic lines now exist for the FLT-antiNodT fusion protein expression construct. These plants are now growing well and cuttings are being taken and rooted to produce multiple vegetatively-propagated plants for each line. This is essential to run multiple tests for the gene expression patterns and HLB resistance levels of each line.
This project aims to understand HLB pathogenesis by analyzing the citrus targets of four effector proteins of Candidatus Liberibacter asiaticus (CLas). Effectors are secreted proteins that perform essential virulence function in bacterial pathogens. They manipulate plant immunity and physiology for the benefit of colonization and disease development. CLas possesses the general Sec secretion system, which secretes about 20-30 effectors into the phloem. We hypothesize that Sec effectors of CLas target key components in citrus, and thereby contributing to HLB. Our bioinformatic and gene expression analyses revealed four Sec effectors of CLas that are highly expressed in infected citrus trees. In this project, we are identifying the direct targets of these effectors in citrus. These targets may play important roles in HLB development and symptom physiology. A major approach that we are using to find the effector targets is yeast two hybrid (Y2H) screen. In the first year of this project, we cloned the four CLas effector genes into an Y2H bait vector, transformed them into the yeast strain AH109. We also constructed a citrus cDNA library using HLB-infected RNA samples. This library was further normalized to exclude the highly abundant transcripts, which may bias the screen. At the end, we obtained a high-quality citrus cDNA library with more than 3 millions of primary clones. In the second year of this project, we completed the Y2H screening using each of the four effectors as the bait respectively. For each effector, more than 10 million yeast clones were screened in three independent experiments. Sequences of potential bait-interacting proteins were determined using next generation Illumina sequencing. These sequences were compared to a non-selected cDNA pool generated from screens using control proteins as the bait. This practice effectively eliminated non-specific interacting proteins. The rest of the sequences represent candidate proteins that specifically associate with a particular effector bait. These sequences have been aligned to the genome sequence of Citrus sinensis to obtain gene identities. After sophisticated statistical analyses, we came up with a list of potential targets for each effector. Our data suggest that one effector specifically targets E3 ubiquitin ligases, which are important enzymes that regulate protein stability. The second effector targets a class of transcription factors that regulate plant immunity. The third effector targets peptidases, which also regulate protein stability. The last effector mainly targets ion-binding proteins. This is interesting because HLB symptoms resemble zinc deficiency, indicating that ion metabolism in citrus is altered by CLas infection. Taken together, we have found promising targets of the four effectors. Our data suggest that the effectors potentially manipulate citrus immunity and metabolism, and thereby contributing to HLB. Our on-going effort is to confirm the interactions between specific baits and their targets using in vitro and in vivo pull-down assays. We are also characterizing the localization of the effectors and the co-localization of effectors with their targets.
This project aims to understand HLB pathogenesis by analyzing the citrus targets of four effector proteins of Candidatus Liberibacter asiaticus (CLas). Effectors are secreted proteins that perform essential virulence function in bacterial pathogens. They manipulate plant immunity and physiology for the benefit of colonization and disease development. CLas possesses the general Sec secretion system, which secretes about 20-30 effectors into the phloem. We hypothesize that Sec effectors of CLas target key components in citrus, and thereby contributing to HLB. Our bioinformatic and gene expression analyses revealed four Sec effectors of CLas that are highly expressed in infected citrus trees. In this project, we are identifying the direct targets of these effectors in citrus. These targets may play important roles in HLB development and symptom physiology. A major approach that we are using to find the effector targets is yeast two hybrid (Y2H) screen. In the first year of this project, we cloned the four CLas effector genes into an Y2H bait vector, transformed them into the yeast strain AH109. We also constructed a citrus cDNA library using HLB-infected RNA samples. This library was further normalized to exclude the highly abundant transcripts, which may bias the screen. At the end, we obtained a high-quality citrus cDNA library with more than 3 millions of primary clones. In the second year of this project, we completed the Y2H screening using each of the four effectors as the bait respectively. For each effector, more than 10 million yeast clones were screened in three independent experiments. Sequences of potential bait-interacting proteins were determined using next generation Illumina sequencing. These sequences were compared to a non-selected cDNA pool generated from screens using control proteins as the bait. This practice effectively eliminated non-specific interacting proteins. The rest of the sequences represent candidate proteins that specifically associate with a particular effector bait. These sequences have been aligned to the genome sequence of Citrus sinensis to obtain gene identities. After sophisticated statistical analyses, we came up with a list of potential targets for each effector. Our data suggest that one effector specifically targets E3 ubiquitin ligases, which are important enzymes that regulate protein stability. The second effector targets a class of transcription factors that regulate plant immunity. The third effector targets peptidases, which also regulate protein stability. The last effector mainly targets ion-binding proteins. This is interesting because HLB symptoms resemble zinc deficiency, indicating that ion metabolism in citrus is altered by CLas infection. Taken together, we have found promising targets of the four effectors. Our data suggest that the effectors potentially manipulate citrus immunity and metabolism, and thereby contributing to HLB. Our on-going effort is to confirm the interactions between specific baits and their targets using in vitro and in vivo pull-down assays. We are also characterizing the localization of the effectors and the co-localization of effectors with their targets.
We have been working to modulate the response of citrus to huanlongbing (HLB) infection, by targeting genes that are up-regulated, and contribute to phloem-plugging in HLB-affected citrus(Albrecht and Bowman, 2008; Folimonova and Achor, 2010; Kim et al., 2009). Our approach has been to use an engineered citrus tristeza virus (CTV) silencing vector (Folimonov et al., 2007; Gowda et al., 2005; Hajeri et al., 2014) to deliver truncated dsRNA sequences homologous to callose (CalS7) and phloem-protein 2 (PP2) implicated in phloem-plugging. These dsRNA molecules are expected to result in the down-regulation or reduced expression of CalS7 and PP2, such that phloem-plugging will be precluded or mitigated in citrus upon infection with HLB. The CTV vector has been engineered to express CalS7 and PP2 truncated sequences, and inoculated into citrus (Citrus macrophylla). Using conventional reverse transcriptase-polymerase chain reaction (RT-PCR), the presence of the engineered virions has been verified in individual citrus plants. Plants positive for the engineered CTV vector were then graft-inoculated into other citrus varieties and tested similarly. The responses of these plants, and appropriate controls, to HLB infection are being tested by exposing young flush to an HLB-positive psyllid colony maintained in the laboratory for four weeks, and following the changes in mRNA expression levels of CalS7 and PP2 via quantitative RT-PCR (qRT-PCR) on a monthly basis. Two-Three months after testing positive for HLB, we have observed changes in CalS7 and PP2 mRNA levels in both HLB-positive treated and non-treated controls. Although the observed changes in PP2-silenced citrus are significantly lower than the non-treated controls, changes in CalS7-silenced plants remain highly variable at this time. We have observed variations in expression of CalS7 and PP2 even in healthy controls, and thus are unable to correlate these changes in expression with HLB-status. It is too early to draw conclusions based on the available data. However, the silenced plants exposed to HLB are beginning to show symptoms of infection, while the microscopy does not show phloem-plugging yet. It is possible that symptom expression is not directly the consequence of phloem plugging. Overall, our experiments are progressing well, and we have discussed our results so far at the just ended International Research Congress on Huanlongbing (IRCHLB IV) in Orlando, Florida. We intend to continue to follow the profile of CalS7 and PP2 expression in experimental plants via qRT-PCR, and to support our data with northern blot analysis to show silencing of CalS7 and PP2, and microscopy to reveal any phloem plugging.
The goal of this project (#894) is to supplement project #707, specifically to determine the efficacy of plant growth regulators (PGRs) as a tool to mitigate declines in citrus tree root and canopy growth resulting from HLB. This project will extend our current work to include detailed greenhouse trials designed to help inform field applications of PGRs on established Hamlin and grapefruit trees in Lake Alfred and the Indian River region. The greenhouse studies will enable us to control environmental variables (soil type, tree age, secondary infections, etc.) that are not possible in the field and develop a fundamental understanding of how PGRs (e.g. 2,4-D, cytokinins, GAs) affect HLB-affected trees compared to healthy trees. Progress in the first quarter included procurement of sixty clean ‘Valencia’ / Kuharske nursery trees and inoculating half of them with HLB by grafting them with shoots from PCR positive trees. At the same time we also non-destructively estimated the root system size of every tree with an electrical procedure that measures root resistance and capacitance. This measurement will serve as the baseline for the current healthy root system status before deterioration from HLB infection begins. As of October 15th, 2014, work is in progress regarding the greenhouse experiments described, above.
Genome of Candidatus Liberibacter asiaticus (CLas) reveals the presence of luxR that encodes LuxR protein, one of the two components typical of bacterial “quorum sensing” or cell-to-cell communication systems. Interestingly, the genome lacks the second components; luxI that produce Acyl-Homoserine Lactone (AHL) suggesting that CLas has a solo LuxR system. We have confirmed the functionality of the CLas solo luxR by constructing a luxR gene promoter fused with a GFP reporter. This has resulted in a functional CLas luxR::GFP monitor strain E. coli similar to that reported by Kock et al (2005). This E.coli strain produces fluorescence if the luxR promoter binds to AHLs or to a eukaryotic signal. Several AHLs, including N-butanoyl homoserine lactone, N-hexanoyl homoserine lactone, N-3-oxo-hexanoyl homoserine lactone, N-3-oxo-octanoyl homoserine lactone and N-3-octanoyl homoserine lactone, as well as extractions from insect and from citrus plant, have been shown to activate CLas luxR. The plant derived extracts are likely to be structurally unrelated AHL mimics. As a response to infection by CLas, citrus may increase the production of its AHL mimic(s), which would bind to LuxR and possibly limit CLas bacterial growth by triggering cell aggregation and consequently limit bacterial movement in planta. The insect extract has a structurally related AHL which may be produced by the endosymbiontic bacteria and bind to CLas LuxR. As a result of this binding, Clas form biofilm on the surface of ACP gut. Currently we are investigating the effect of many compounds known to be signals for bacterial LuxR, especially those found in citrus phloem sap, on the activity of CLas LuxR. These compounds include, but not restricted to Indole-3-acetic acid (IAA), indole, .-amino butyric acid (GABA), salicylic acid (SA), Riboflavin and Lumichrome. We expressed CLas LuxR in citrus using the CTV-based vector system. LuxR expressing citrus showed equally distributed severe symptoms when infected with CLas. Rearing infected ACP on healthy LuxR plants resulted in a diminishing CLas population. Interfering with CLas cell-to-cell signaling may lead to new avenue of control strategies.
The general goal of this project is to rapidly propagate complex citrus rootstock material for field testing. The rootstock materials to be tested will be products of the Citrus Improvement Program at the UF-IFAS-CREC in Lake Alfred. Specifically, these materials will be selected based upon their performance in the ‘HLB gauntlet’: Promising rootstock genotypes will have already been evaluated in the greenhouse and field for their ability to grow-off citrus scions that have been exposed to CLas-positive budwood and CLas-positive Asian citrus psyllids. Once candidate rootstock materials have successfully passed through this gauntlet, they will be propagated via rooted cuttings en masse in a psyllid-free greenhouse at the UF-IFAS-IRREC in Fort Pierce. From there, rootstock materials will be budded with scion materials and planted in the field for further testing for their long-term performance. The start date for this project was April, 2013. To date, the progress of this project is as follows: – Two (2) misting chambers to propagate candidate, rootstock materials as rooted-cuttings have been constructed. – Propagation materials (containers, soilless media, and rooting hormones) have been purchased. – Funds from this project were used to support the construction of a new greenhouse at the IRREC. This greenhouse is completed and operational. – The first cohort of advanced, tetratzygous citrus rootstock materials for en masse propagation are currently being propagated. – The second cohort of advanced, tetrazygous citrus rootstock materials for en masse propagation have been identified and are being prepared to have cuttings taken from them. – In addition to the 1st & 2nd cohorts of tetrazygous rootstocks, promosing diploid rootstocks have also been identified and are being prepared to have cuttings taken from them. As of January 2015, plants are still growing in the green house and no significant changes to report.
The goal of this project (#894) is to supplement project #707, specifically to determine the efficacy of plant growth regulators (PGRs) as a tool to mitigate declines in citrus tree root and canopy growth resulting from HLB. This project will extend our current work to include detailed greenhouse trials designed to help inform field applications of PGRs on established Hamlin and grapefruit trees in Lake Alfred and the Indian River region. The greenhouse studies will enable us to control environmental variables (soil type, tree age, secondary infections, etc.) that are not possible in the field and develop a fundamental understanding of how PGRs (e.g. 2,4-D, cytokinins, GAs) affect HLB-affected trees compared to healthy trees. Progress in the first quarter included procurement of sixty clean ‘Valencia’ / Kuharske nursery trees and inoculating half of them with HLB by grafting them with shoots from PCR positive trees. At the same time we also non-destructively estimated the root system size of every tree with an electrical procedure that measures root resistance and capacitance. This measurement will serve as the baseline for the current healthy root system status before deterioration from HLB infection begins. As of January 15th, 2015, work is in progress regarding the greenhouse experiments described, above.
Our project aims to provide durable long term resistance to Diaprepes using a plant based insecticidal transgene approach. In this quarter, as proof of concept to determine the root specific nature of the promoters (RB7, C1867 or SLREO), we have incorporated the promoter-gus sequences into N. benthamiana and Carrizo citrange and several plantlets have been regenerated. Testing of these plants to confirm the root specific activity of our promoters will be performed as they become available. In addition, we have initiated experiments to incorporate the plant transformation vectors containing the GNA, APA and ASAL genes driven by either the root specific RB7 promoter or the citrus derived C1867 promoter into Carrizo citrange. Stacked constructs, each containing the GNA, APA or ASAL genes with the CpTI gene driven by the SLREO promoter have been produced and are also being incorporated into Carrizo citrange.
In order to better understanding of the transmission mechanism of citrus Huanglongbing (HLB) by the insect vector, Asian citrus psyllid (ACP), we have been working on unraveling the protein-protein interactions (PPI) between ACP and the HLB associated bacterial agent, Candidatus Liberibacter asiaticus (CLas) by means of proteomics. Complexome is the whole set of the protein-protein interactions in a particular cell or organism. During the transmission process, CLas bacteria traverse inside the insect vector systemically and various PPIs and protein complexes (formed by protein constituents from both CLas and ACP) must be involved; therefore, our aim was to find the proteins involved in the CLas-ACP interactions. Several approaches have been used including protein-overlay assay (Far-Western) and Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) 1-Far-Western: we used phloem sap from infected citrus as source for CLas. The phloem sap was used to overlay ACP proteins that were separated by SDS-PAGE and trans-blotted onto PVDF membrane. The complexes were revealed using CLas-specific antibodies. We identified V-ATPase, ATPase-TER 94, beta-tubulin, and actin as binding proteins for CLas. 2- BN-PAGE We have established the BN-PAGE system in our lab to study the protein complexes (i.e. complexome) from CLas-free and CLas-infected ACP in their native status, and a further separation of the protein complexes in BN-PAGE by a second dimension of SDS-PAGE which helped us obtain more detailed information on all the subunits or constituents of the protein complexes at their denatured status. We have successfully located several protein spot candidates for protein identification by mass spectrometry. Among the identified proteins, ferritin subunits and transferritin formed complexes with CLas proteins. The identified proteins should be a good candidates for RNAi technology using CTV-based vector. Expression of dsRNA against those protein in citrus may reduce or block the transmission of CLas by its vector ACP.
The project has two objectives: (1) Increase citrus disease resistance by activating the NAD+-mediated defense-signaling pathway. (2) Engineer non-host resistance in citrus to control citrus canker and HLB. For objective 1, we have repeated NAD+ treatment experiment. Again, both soil drench and foliar spraying of NAD+ have been conducted. The plant defense activator Actogard, which is highly effective against citrus canker was included in the experiment as a control. We found that while foliar spraying did not provide significant protection against citrus canker, soil drench induced strong resistance against the pathogen. Interestingly, we observed strong systemic protection against canker by NAD+ one month after the treatment in upper new flushes. We are planning to repeat the experiment to confirm the systemic effects. Meanwhile, we are still trying to find the best approach for NAD+ application. NAD+ analogs are under test for identifying potential chemicals to control citrus canker. For objective 2, 30 transgenic lines expressing ELP3 and 22 lines expressing ELP4 have been generated. The transgenic lines have been molecularly characterized to confirm the presence and expression of the transgenes. The transgenic plants are growing in greenhouse and will be tested for canker resistance. Citrus homologs of ELP3 and ELP4 have been cloned and sequenced. We are cloning the two genes into T-DNA vector and will be transformed into the Arabidopsis elp3 and elp4 mutants, respectively, to confirm their functionality.
The project has two objectives: (1) Increase citrus disease resistance by activating the NAD+-mediated defense-signaling pathway. (2) Engineer non-host resistance in citrus to control citrus canker and HLB. For objective 1, we have repeated NAD+ treatment experiment. Again, both soil drench and foliar spraying of NAD+ have been conducted. The plant defense activator Actogard, which is highly effective against citrus canker was included in the experiment as a control. We found that while foliar spraying did not provide significant protection against citrus canker, soil drench induced strong resistance against the pathogen. Interestingly, we observed strong systemic protection against canker by NAD+ one month after the treatment in upper new flushes. We are planning to repeat the experiment to confirm the systemic effects. Meanwhile, we are still trying to find the best approach for NAD+ application. NAD+ analogs are under test for identifying potential chemicals to control citrus canker. For objective 2, 30 transgenic lines expressing ELP3 and 22 lines expressing ELP4 have been generated. The transgenic lines have been molecularly characterized to confirm the presence and expression of the transgenes. The transgenic plants are growing in greenhouse and will be tested for canker resistance. Citrus homologs of ELP3 and ELP4 have been cloned and sequenced. We are cloning the two genes into T-DNA vector and will be transformed into the Arabidopsis elp3 and elp4 mutants, respectively, to confirm their functionality.
Seasonal root sampling continues in two field sites for root density and root growth. We have completed a second year of root growth data from Hamlin/Swingle and are continuing to collect a second year of root growth data on Valencia/Swingle. Results continue to emphasize the need to use treatments that improve root longevity as the main method of managing HLB root loss. Additional root growth appears to occur at the expense of older roots and is unlikely to provide sustained improvement in root density. Root tube images taken with the root scanner to assess root growth, lifespan, and death continue to be collected monthly. Visual observation shows that the method is working well, but data analysis has been delayed due to an unexpected software glitch, a new version has been released and we are working through a backlog of images. Only one rootstock tested to date has shown a significant difference in response to HLB. It remains the only rootstock with significantly better root density (increased) when infected by HLB. Root loss has not been observed yet in this rootstock. Fruit drop data has been collected to determine if the increased root density is associated with reduced fruit drop. We continue to monitor the most promising rootstocks identified in the field trial to HLB using rhizotrons in the greenhouse. The second set of rootstocks has been grafted with Hamlin scion or left ungrafted to monitor the effects of the scion rootstock interaction on the rootstock response. Phytohormone analysis has been completed on roots, but no clear consistent change has been observed in old roots or new root tips. Microscopy of the root samples to identify the mechansim of root dieback is awaiting the arrival of a new confocal microscope.
We monitored stem water potential using thermocouple psychrometers on trees in a commercial Hamlin grove in Lake Alfred (Gapway Grove) that has an established experiment comparing Advanced Citrus Production System (ACPS) and conventional grower practices (C). We found no statistical difference in the mean minimum daily water potential between the two treatments during the three sampling periods between April 4-10, April 30-May 9th, May 21-May 23, however we did find greater drought stress in ACPS trees on the hot days in May. There was a borderline statistical difference (p=0.065) in the slope of the water potential curves, where trees in the conventional grower irrigation treatment did not experience as rapid of an increase in drought stress compared to the ACPS trees. These data support xylem vulnerability curves generated in the lab that estimate the loss of hydraulic conductivity resulting from drought stress, where the ACPS trees suffered greater losses in conductivity during moderate drought conditions. We found no significant differences in xylem vessel diameters, but we did note that the pit membranes that prevent losses in hydraulic conductivity were significantly thicker in the C trees compared to ACPS trees. Thus, we found that ACPS trees were more vulnerable to the effects of drought stress on a daily basis during the early part of the growing season, presumably due to the fact that they receive more regular irrigation and have adapted to a wetter, more consistent soil moisture environment, and have not invested in additional resistance to the effects of drought compared to the C trees that are irrigated on a less frequent basis. This study highlights the importance of maintaining consistent irrigation for field grown trees, and that clogged or damaged irrigation systems in ACPS trees are a significant risk. A peer-reviewed publication with these data is currently in progress for submission to Tree Physiology. A second component of this study was to determine whether plant growth regulators (PGRs) could be used in a heavily infected Hamlin grove in Lake Alfred to mitigate preharvest fruit drop. Our trial focused on replicating data from colleagues in Brazil who reported benefits of using frequent, low doses of 2, 4-D throughout the growing season. We monitored fruit drop every two weeks starting in September through harvest in December, and found no significant differences between the different treatments. HLB symptoms were somewhat variable in this block, and we saw several mature trees die during this single season, but our study included enough trees to control for tree health. In some cases, applying the commercially-available adjuvant product EcoAgra showed a slight decrease in preharvest fruit drop, but there was enough variability in the data to suggest that none of the treatments we applied to these Hamlin trees was sufficient to reduce drop. At this point we do not recommend 2,4-D at the concentration and frequency applied in this study to be used to mitigate preharvest fruit drop, but these data should be considered within the scope of other PGR trials occurring simultaneously around the state. Symptom severity, local climate, soil differences, and existing pest or disease issues could account for success in some areas but not others.
This is a continuing project to find economical approaches to citrus production in the presence of Huanglongbing (HLB). We are developing trees to be resistant or tolerant to the disease or to effectively repel the psyllid. First, we are attempting to identify genes that when expressed in citrus will control the greening bacterium or the psyllid. Secondly, we will express those genes in citrus. We are using two approaches. For the long term, these genes are being expressed in transgenic trees. However, because transgenic trees likely will not be available soon enough, we have developed the CTV vector as an interim approach to allow the industry to survive until resistant or tolerant trees are available. A major goal is to develop approaches that will allow young trees in the presence of HLB inoculum to grow to profitability. We also are using the CTV vector to express anti-HLB genes to treat trees in the field already infected with HLB. At this time we are continuing to screen possible peptide candidates in our psyllid containment room. We are now screening about 80 different genes or sequences for activity against HLB. We are starting to test the effect of two peptides or sequences in combination. We have developed methods to be able to screen genes faster. Finally, we have found a few peptides that protect plants under the high disease pressure in our containment room with large numbers of infected psyllids. We now are examine combinations of peptides for more activity. We recently examined all of the peptides constructs for stability. The earliest constructs have been in plants for about nine years. Almost all of the constructs still retain the peptide sequences. One of the peptides in the field test remained stable for four years. All of these constructs had the peptide gene inserted between the coat protein genes, which is positioned sixth from the 3′ terminus. However, we have found that much more foreign protein can be made from genes positioned nearer the 3′ terminus. Based on that we built constructs with the peptide gene next to the 3′ terminus. These constructs produced much greater amounts of peptide and provided more tolerance to Las. Unfortunately, they are less stable. So now we are rebuilding constructs with the peptide gene inserted at an intermediate site hoping for a better compromise of amounts of production and stability. We are screening a large number of transgenic plants in collaboration with Dr. Zhonglin Mou, Department of Microbiology and Cell Science in Gainesville, to test transgenic plants over-expressing plant defense genes. We have found that three different lines appear to be giving strong tolerance against HLB. We are propagating the plants for more extensive analysis.
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