This project aims to understand important components during HLB pathogenesis. Our previous research identified four secreted proteins, called effectors, from the HLB-associated bacterium Candidatus Liberibacter asiaticus (Las). Effectors are known to play important virulence function in microbial pathogens. Our goal is to characterize the targets of these effectors in citrus. This project will advance our understanding on the basic biology of HLB pathogenesis and facilitate the development of control strategies. Our research has been focusing on four Las effectors that are highly expressed in HLB-infected citrus trees. We hypothesized that their targets in citrus contribute to Las infection and/or symptom development. The main approach we are using to identify the effector targets is yeast two hybrid (Y2H) screening. In the past two years of this project, we accomplished the following experiments: 1) expression analysis of the Las effectors; 2) cloning and expression of the effector genes in yeast; 3) construction of a normalized citrus cDNA library with more than 3 millions of primary clones using HLB-infected RNA samples; 4) Illumina sequencing-based Y2H screening using each of the four Las effectors as the bait and sequencing data analysis; 5) subcellular localization analysis of the Las effectors. At the beginning of the third year of this project, we focused on experimental confirmation of effector targets. From Y2H screening, multiple potential targets were identified for each effector. We performed extensive literature search and prioritized our effort on candidates with potential roles in plant defense or HLB symptom development. These candidates were re-cloned from citrus cDNA and were subsequently examined for their interaction with the corresponding effector by targeted Y2H. To this end, we have confirmed the interaction partner of two Las effectors. These two effectors are expressed more than 10 folds higher in citrus than in psyllids, suggesting that they may play a role in HLB pathogenesis. One of these two effectors specifically targets E3 ubiquitin ligases, which are important enzymes that regulate protein stability and are also well-known virulence targets of other bacterial and fungal pathogens. The other effector targets ion-binding proteins that are important for ion uptake and photosynthesis. Our on-going effort is to finish the in vitro and in vivo co-immunoprecipitation assays to further confirm the effector-target interaction. We will also determine the co-localization of effectors with their targets using confocal microscopy analysis.
The driving force for this project was the need to evaluate citrus transformed to express proteins that might mitigate HLB, which required citrus be inoculated with CLas. Although citrus can be manually inoculated by grafting with infected budwood, a program using CLas-infected ACP was preferred primarily because this is what occurs in nature. Nine colonies of infected ACP for inoculations were initially established in a walk-in chamber. Additional colonies of infected ACP were later established in two small greenhouses and in a second walk-in chamber. For each individual colony, a potted lemon or citron tree graft-inoculated with CLas (testing PCR-positive and showing HLB symptoms) was placed into a cage and trimmed to stimulate flush; ACP were introduced and allowed to reproduce; and the colonies were maintained over time by regularly trimming plants and introducing additional ACP as needed. The source of CLas for these lemon and citron trees was the original HLB-infected tree found during 2005 in south Florida. Germplasm to be challenged for HLB resistance was supplied by USDA-ARS citrus breeders. To inoculate plants, individual plants were caged for 2 wk with 20 psyllids from one of the infected colonies, after which the plants were held together for six months in a greenhouse with an open infestation of infected psyllids. After this two-step inoculation procedure, the plants were returned to the breeders. Challenges associated with the program have included variability in percentages of ACP testing CLas-positive in a single colony over time and among different colonies at any given time. Consequently for the first inoculation step we always used ACP from colonies with the highest percentages of infected ACP, and we increased the number of colonies to increase numbers having high percentages of infected ACP. Lots of qPCR assays were required to monitor infected ACP colonies and infected source plants. Growth chamber colonies generally have had greater percentages of infected ACP than greenhouse colonies, probably due to more stable environmental conditions in chambers. Another challenge has been that, among ACP that test PCR-positive for CLas, relatively low transmission rates have been reported (e.g., 12% or less). Therefore, caging an individual plant with 20 psyllids from an infected colony may not always result in inoculation. Success of the first inoculation step would likely be increased by increasing the number of ACP per plant. Subsequently holding the plants for six months in the greenhouse with free-roaming infected psyllids increases the probability of inoculation. With respect to the second inoculation step, population levels of ACP in the greenhouse were sometimes severely reduced as a consequence of damage to oviposition sites by western flower thrips or spider mites. Also, sometimes population levels of ACP eggs and nymphs were severely reduced by western flower thrips, and further reductions in populations of nymphs were sometimes caused by Tamarixia parasitoids that invaded the open infestation of ACP. Effectiveness of the inoculation program has been slow to gauge. A series of experiments was initiated during 2014 specifically to evaluate inoculation success. Meanwhile, recent feedback from inoculations of rootstock material gave some insight. Eleven groups of rootstock material (3,105 plants total) were passed through the inoculation program during 2011-2014. The average percentage of infected ACP used in step one was 52% (range 24 to 80%). qPCR 12 to 19 months after the two-step inoculation process indicated an average of 62% success in inoculating citrus. Plants that escaped inoculation had another opportunity for inoculation after being transplanted to the field.
Core Citrus Transformation Facility (CCTF) continued to operate at the high level it did in the previous period and produced transgenic material in a timely and dependable manner. CCTF maintained its standing as reputable partner that delivers transgenic material in reasonable amount of time and continued to be sought for services. This quarter marks the end of three year period that saw significant but expected increase in number of orders. With six new orders received most recently, CCTF has just surpassed its 200th order and 96 of those were placed within the last three years. Clients requested transgenic Duncan grapefruit and Valencia orange in the newest orders. The number of plants produced in this quarter is 85 bringing a total to 730 plants for the duration of the funding period. Most of the produced plants were of Duncan grapefruit cultivar, followed by Carrizo rootstock, Valencia orange, some Citrus macrophylla, Swingle citrumelo, and Mexican lime. This strong bias towards Duncan grapefruit points towards need of researchers to get fast answers regarding candidate gene that could potentially render Citrus plants tolerant/resistant to diseases. Since Duncan grapefruit is highly susceptible to both Huanglongbing (HLB) and citrus canker, challenging transgenic plants expressing gene of interest with bacteria causing these two diseases will quickly reveal if that gene holds any promise or not. Throughout the duration of this project, CCTF processed about 400000 explants. Approximately 5% of explants were lost to contamination which brings the total number of explants producing data to 380000. Overall transformation efficiency expressed as the percentage of positive shoots out of all of those tested was about 3% which is low. One of the reasons for this is the nature of orders received. Amongst 96 received orders, there were 34 for which total of just a few transgenic plants were produced. This was a result of withdrawal of 10 orders, presence of sequence detrimental for shoot development in binary vector used for transformation in six orders, and for 18 orders transformation was either not possible or achieved at the very low rate. The second reason for low transformation rate is the quality of seeds/seedlings resulting from the effect HLB infection has on fruit growing on trees. In the immediate future, CCTF will dedicate increasing efforts to acquire fruit/seeds of higher quality than what it was used recently. The labor force and the income of CCTF remained relatively stable during the last three years and changes in facility s staff never compromised the level of operation. Number of orders presently serviced by the CCTF and those that were either announced or anticipated in the near future is clear indication of sustaining interest of researchers in transgenic citrus plants. They also assure CCTF will stay busy for the years to come.
The original and initial intent of this one-year-funded project was to supplement CRDF-CATP #707 by developing greenhouse and expanded field trials exploring the potential efficacy of 2,4-D applications to reduce citrus fruit drop and its effects on overall tree health. Potted Valencia trees were purchased from a commercial citrus nursery and half were bud-inoculated with CLas to determine the effects of 2,4-D applications on CLas+ trees in a controlled greenhouse environment. The project duration has since expired and funding no cost extension was not granted. It is therefore not presently clear as to the potential efficacy of 2,4-D in reducing citrus fruit drop.
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 have produced a large amount of inoculum for a large field test via Southern Gardens Citrus. 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 are propagating a progeny set of plants of the promising candidates for a final greenhouse test.
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 have produced a large amount of inoculum for a large field test via Southern Gardens Citrus. 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 are propagating a progeny set of plants of the promising candidates for a final greenhouse test.
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 several plantlets have been regenerated. These constructs were also incorporated into Carrizo citrange in the previous quarter, but we have had a severe Mould mite infestation in our tissue culture room which destroyed most the promoter-gus citrus plantlets. A repeat experiment to produce fresh carrizo citrange plants expressing the root specific promoters were carried out in this quarter and plantlets are being regenerated. The Carrizo citrange plants expressing a root specific promoter – insecticidal gene construct (GNA, APA or ASAL genes individually or stacked with the CpTI gene) were not affected by the mite infestation and several plants have been hardened and transferred to the greenhouse for growth.
The goal of this project was to create canker-resistant citrus through a strategy of an engineered cell death response to Xanthomonas citri pv. citri TAL effectors. During the project, we defined sequences of 14 distinct effector binding elements (EBE) recognized by Xcc TAL effectors. Constructs were created that incorporated these EBEs into the Bs3 promoter driving a pathogen effector gene (either avrGf1 or avrGf2) that triggers a hypersensitive cell death reaction in several citrus cultivars. Two key constructs were one with all 14 binding sites (14EBE) and one with four binding sites (4EBE) that was expected to be bound by at least two TAL effectors from each X. citri strain characterized. These constructs were shown to function as expected in a transient assay in citrus leaves: they produced a hypersensitive response and reduced bacterial growth when co-inoculated with a Xanthomonas strain carrying a TAL effector recognizing one of the EBEs. In the absence of pathogen inoculation, the promoters are tightly “off” in the transient assay. However, despite the tight regulation of these constructs in a transient assay and multiple attempts to produce stable transformants in Duncan grapefruit or sweet orange, we have been unable to recover transgenic lines. We also tested Carrizo citrange, a citrus variety that does not exhibit a hypersensitive response to AvrGf1 or AvrGf2, and we were able to recover transformants. These results suggest cryptic induction of the construct at some point during the transformation process, causing transformants to be selected against. A second promoter derived from the citrus Lateral Organ Boundaries (LOB) gene also failed to produce transformants when driving AvrGf2 expression. Therefore, despite the theoretical promise of the approach, more work would be needed to define a promoter and executor gene combination that could be efficacious in citrus.
Seasonal root sampling continues in two field sites for root density and root growth. We are collecting a second year of root growth data from Hamlin/Swingle and have 1 year of root growth data on Valencia/Swingle. Results so far emphasize the need to use treatments that improve root longevity as the main method of managing HLB root loss. Root growth stimulation is unlikely to improve root density. Preliminary tests of root tubes are almost complete. This will allow for more rapid quantification of root growth and death with less damage to observed trees. Field site selection for the first set of tubes is underway. Sampling at a rootstock trial site continues. Only one rootstock tested to date has shown a significant difference in response to HLB we will be installing root tubes to compare root growth and root death data to rhizotron experiments. We continue to monitor the most promising rootstocks identified in the field trial to HLB using rhizotrons in the greenhouse. The first experiment is nearing completion and data analysis of root growth and root longevity in response to Las is currently underway. The analysis is taking longer than expected because of some variations introduced by the original setup that need to be corrected for. Fine tuning is being done to the setup to reduce the data analysis steps in future experiments. Additional rootstocks are being considered for the second round of rhizotron experiments and should be planted and inoculated shortly. Method development to characterize the mechanism by which Liberibacter causes root death is underway and the experimental samples will be collected shortly.
To confirm that treatments with acidified irrigation water reduce the impact of bicarbonate stress on root health, we surveyed 8 ridge groves in Highlands county and 4 flatwoods groves in Hardee county with high bicarbonate stress as detected in our 2013 survey. All the blocks are less than 10 year old Valencia trees on bicarbonate sensitive rootstocks, Swingle and Carrizo. The survey is bimonthly to follow the recovery of these blocks and at harvest to compare 2014 season block yields 1.0 to 1.5 years after acid treatments began. From May to November root density fluctuated but was maintained at about the same level throughout the season. Root density was 5-10X higher in the ridge than in the flatwoods groves. Phytophthora populations were low or non-detectable in ridge groves but were always present and about 2-3x higher in the flatwoods groves. Soil pH ranged from 5.0-6.0 in the ridge after 1-1.5 years of acidification. pH in the flatwoods groves was initially in this range but rose above 6.5 and remained at that level. Flatwoods groves with higher bicarbonates in irrigation wells and soil require higher rates of acidification treatment to reduce below 6.5 as recommended for Swingle and Carrizo rootstock groves. Block yields will be evaluated for response in production compared to previous seasons. Surveys are continuing in 2015 with the addition of 4 groves without acidification in Highlands and Hardee counties to serve for comparison with an un-managed check. Recommendations based on these results were published in the May 2015 issue of Citrus Industry magazine.
Recent evaluation of root production on HLB-affected trees compared to presumed healthy trees confirms that root loss is due to reduced root longevity. This root loss is exacerbated by biotic and abiotic stresses in the rhizosphere. Increased susceptibility of Las’infected roots to Phytophthora spp. is evidenced by statewide populations that have fluctuated from unprecedented highs in the 2011 season to an unprecedented low in 2013 compared to 25 years of pre-HLB soil populations. Phytophthora propagules per soil volume and per root resurged in 2014 in response to a more than doubling of root density based on intensive (i.e., local repeated measures) and extensive (i.e., Syngenta statewide survey) sampling compared to 2013. Substantially higher root density in Florida groves in spring and summer 2014 has so far this harvest resulted in less fruit drop than in the previous two seasons. Both field surveys and greenhouse experiments show that HLB increases the susceptibility of the citrus root system to the damaging root pathogen P. nicotianae. Increased P. nicotianae infection suggests that root system damage occurs more quickly in the presence of both pathogens. Chemical control of P. nicotianae slows infection of the root system and may slow yield decline in HLB-affected trees in Phytophthora-infested groves, however, HLB reduces the effectiveness of fungicides for control of Phytophthora root rot as a consequence of increased root susceptibility to P. nicotianae infection.
For 2015 season, FireWall 50WP (50% streptomycin; Agrosource, Inc.) has been granted an EPA section 18 registration for control of citrus canker in Florida grapefruit. The label for FireWall restricts use to no more than two applications per season. As a condition for FireWall registration, EPA requires monitoring of Xanthomonas citri subsp. citri (Xcc) for streptomycin resistance in treated groves. The objective of this survey is to apply our published protocol (Behlau et al., 2012) for sampling canker-infected grapefruit leaves for isolation and detection of streptomycin resistant Xcc. The survey for 2015 season will be conducted in November 2015 and the report of results submitted to FDACS. Greenhouse trials to measure the residual systemic activity of streptomycin against Xcc in leaves after foliar spray will be repeated as in 2014 to confirm trans-cuticular and upward movement of streptomycin into new foliage via the xylem.
Objective 1. As reported previously, methyl accepting chemotaxis proteins (MCPs) were identified for wide (Xcc62, Xcc306) and narrow host range strains of Xanthomonas citri subsp. citri (Xcc) compared to X. fuscans subsp. aurantifolii B and C strains, X. alfalfae sbsp. citrumelonis (Xac) or X. campestris pv. campestris (Xc). All Xcc strains showed similar MCP content except Xcc306 which lacked some of the MCPs. MCP analysis was expanded to include Xcc strains from different geographical areas. 37 strains from Argentina (27%), Uruguay (32.5%), Brazil (27%) and 5 isolates from citrus of uncertain origin (13.5%) were analyzed for 15 MCPs. Only one of the MCPs was conserved among all Xanthomonas strains (XCV1951), and two of them were specific of Xc (XCC0324 and XCC1954). Objective 2. The role of extracellular DNA (eDNA) was evaluated in the formation and stabilization of the biofilm matrix. The presence of eDNA in biofilm was confirmed for several Xcc strains and Xac. DNAse treatments of Xcc strains and Xac reduced biofilm formation at the initial stage of development as well as disrupted preformed biofilm. The DNAse effect on formation or disruption varied among Xcc strains and Xanthomonas species and indicates differing roles for eDNA. Differences in fiber structures containing eDNA in biofilms, bacterial cultures, and in twitching motility assays were detected by SYTO-9 staining and fluorescence microscopy. The proposed roles for eDNA in the early stages of biofilm formation are as an adhesin and in mature biofilms as a structural component. .
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. At Penn State, all the transgenic lines are being propagated as vegetative cuttings. We have achieved over 50% success rate in rooting cuttings from the plants, and rooted cuttings are now growing for all of the independent lines. The rooting process for cuttings takes 3-4 months in our facility at Penn State. We have performed two protein immunoblots with an antibody that will detect the FLT-antiNodT fusion protein (anti-cmyc antibody). We detected protein of the correct size (~50 kD) as a single band without degradation products in two different lines, and not in control plant samples. These results are very promising and are consistent with successful expression of full-length FLT-antiNodT fusion protein in ‘Duncan’ grapefruit. During the next quarter, we will be repeating these experiments with all of the transgenic lines and using improved protein extraction techniques to develop better western blot images and documentation.
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 continued optimizing the NAD+ treatment conditions. Both soil drench and foliar spraying of NAD+ were tested again. Foliar spraying did not provide significant protection against citrus canker, soil drench induced strong resistance against the pathogen. We previously observed strong systemic protection against canker by NAD+ one month after the treatment in upper new flushes and are repeating the experiment to confirm the systemic effects. We are also testing different recipes for stabilizing NAD+ on the surface of leaves or in soil, since this chemical is not stable under aqueous conditions. For objective 2, transgenic citrus plants expressing the Arabidopsis nonhost resistance genes are growing in greenhouse and will be tested for canker resistance. We are also propagating the transgenic plants to produce progenies for HLB resistance test. Citrus homologs of the Arabidopsis nonhost resistance genes have been cloned and sequenced. To test their functionality, we have cloned the citrus homologs into plant expression vector and have transformed into the corresponding Arabidopsis mutants. T1 plants are growing.
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