The one year study of the in vivo tracking of FT1, FT2, and FT3 in various citrus trees differing in age and phenotype is concluded and is being analyzed. A study of CiFT3 transgenic plants treated with various growth regulators has been performed and all of the data have been collected except for the flowering dates of the nontransgenic control plants that have not yet flowered (they should soon). The growth hormones produced striking and individually different phenotypes in each treatment. The endogenous ciFT3 promoter was successfully cloned to be used in the transcription activator-like (TAL) effector system inducible by methoxyfenozide that will hopefully activate the naturally present FT3 gene in citrus. The complete construct is completed and is being tested in tobacco for a rapid test before citrus experiments are started. This research will be presented at the upcoming ASHS national meeting.
The main objective of this research was to study the CLa-flg22 triggered immune response (basal defense or PTI) in citrus genotypes with different levels of tolerance to HLB and the effect of chemicals associated with SAR priming and signaling (azelaic acid and salicylic acid) have on this response and determine if they could be used to enhance the tolerance against this important pathogen. We analyzed the response of ‘Duncan’ grapefruit (considered susceptible to HLB) and ‘Sun Chu Sha’ mandarin (considered moderately tolerant to HLB) after inoculation with Candidatus Liberibacter asiaticus flagellin 22 (CLas-flg22) using comparative Ct real time PCR. The expression of 16 defense-associated genes was determined. In ‘Sun Chu Sha’ we observed that PBS1, NDR1, RAR1, SGT1, EDS1, EDR1, PAL1, AZI1, NPR2, NPR3 and RdRp were significantly induced compared to water controls. However, in ‘Duncan’ only PR1 was significantly induced compared to water controls. Our results suggested that PTI (PAMP-triggered immunity) was induced by CLas-flg22 in the more tolerant genotype but not the susceptible one. These results were presented during the Third International Research Conference on HLB held in Orlando, Florida on February 4-8 of 2013. Additionally, although not part of the original proposal, RNAseq samples were generated from ‘Sun Chu Sha’, ‘Nagami’, and ‘Duncan’, each treated with CLas-flg22 plus controls to further characterize gene expression in the different citrus types beyond the scope that could be analyzed using real time PCR. The use of RNASeq increased and the cost of this technique came down after we wrote the proposal and we felt it would generate important information and verify our results. The sequencing has been finished and we have analyzed the results partially; however, full analysis is still in progress and has not been completed yet. Regarding the chemical applications, we did not observe an increased immune response to CLas-flg22 by pre-treating with azelaic acid (in terms of heightened defense gene expression). However applications of salicylic acid did enhance the response of some defense genes. Subsequently ‘Sun Chu Sha’, ‘Nagami’ and ‘Duncan’ plants were pre-treated with CLas-flg22 peptide 24 hours prior to inoculation with Xanthomonas citri pv citri (Xcc) in an attempt to corroborate whether CLas-flg22 was capable of inducing PAMP-triggered immunity (PTI). The Xcc population/concentration in the inoculated leaves was determined via colony forming units (cfu) by a standard procedure in a time course of up to 2 days post inoculation (dpi). Bacterial growth in the CLas-flg22 pretreated leaves was lower than in those treated with water in both ‘Nagami’ and ‘Sun Chu Sha’ but not in ‘Duncan’, confirming that PTI was triggered. We used Xcc instead of CLas because of the impossibility of infiltrating the latter pathogen and shorter times to estimate the results. Similar experiments using Xcc-flg22 also triggered PTI in resistant genotypes but not susceptible ones. Taken together our results are encouraging and exciting: CLAs-flg22 induces PTI and the level of induction correlates with the levels of resistance observed in different genotypes. Thus, stronger induction of PTI could potentially lead to tolerant or more resistant genotypes to both HLB and citrus canker. A manuscript describing this research has been submitted for publication.
The objectives of this project are to characterize the molecular interactions between the effectors and the host mitochondrial proteins; to screen for molecules that inhibit the effector functions; and to control HLB using the inhibitor(s) and/or other related molecules. To understand the function(s) of LasA1 and LasA2, we have made several constructs in Gateway’ pDONR’ Vector, and pGWB expression vectors, which contain different versions of the lasA1 and lasA2 genes. In addition, we have made several constructs for development of transgenic citrus via Agrobacterium-mediated transformation. We are analyzing these constructs for their transient expression in Nicotiana benthamiana and stable expression in transgenic Arabidopsis thaliana and citrus. We have obtained transgenic lines with these constructs. These transgenic Arabidopsis lines were verified by PCR and RT-PCR and their segregation in T2 and T3 were analyzed. Arabidopsis expressing LasA1-PFLAG showed a retarded growth and/or overgrowth of their roots. Moreover, the leaves displayed different shape with white-silver dechlorophyllation compared to the the wild type, while Arabidopsis lines expressing LasA2-PFLAG showed similar abnormal phenotype with less severity but normal root growth. We also expressed LasA1 protein using the Champion’ pET Expression System containing a polyhistidine (6xHis) tag in E. coli. Purified LasA1 protein are used for antibody production and crystallization study. Immunoprecipitation and elution of FLAG-tagged autotransporters from Agro-infiltration in Nicotiana benthamiana yielded several protein candidates, indicating LasA1/LasA2 interacted with mitochondria and chloroplast proteins. To further confirm their interactions, constructs for yeast two hybridization assay have been made. The antibody against LasA1 protein are able to detect LasA1 protein from transient-expressed LasA1 and Las-infected plant tissue using Western blot. The successful generation of this antibody will enhance our research in several aspects. Meanwhile LasA1 sequence has been optimized for a variety of parameters that are critical for efficient gene expression in plants. 30 destabilizing elements, 2 potential destabilization sequences and 2 cryptic splice sites were found and optimized. Transient expression of the optimized lasA1gene yielded much higher level of expression. In addition, another hypothetical protein has been expressed in planta via transient and stable transformation, and founded to affect host resistance to a bacterial pathogen. The antibody against this protein is able to detect this antigen both in the transgenic plants and in the Las-infected plants. Meanwhile, the Western blot results revealed unique formation of this protein in E. coli and plants. Citrus plants expressing this protein displayed HLB-like symptom, yellow shoot. Further characterization of this effector and identification of factors affecting its stability in plants is underway.
The objective of this research will 1) characterize Pr-D (FP3) and its role and disease suppression; 2) investigate the dynamics of the prophages/phages in Las bacteria by revealing the variations in gene expression and recombination; and 3) identify critical elements, such as heat and chemical stress that facilitates lytic activities of the prophages. In addition, we will demonstrate whether or not if the ‘cross protection’ using mild strains of Las bacteria will work for the HLB pathosystem along with quantitative detection protocols for prophage-based strain differentiation. We have propagated more Las-infected periwinkle and citrus plants that contain high titers of prophage/phage FP3, which will be used for isolation and characterization of prophage/phage FP3. Different varieties of citrus plants inoculated with a mild strain have been evaluated in greenhouse. Intriguingly, different varieties showed different response to the “mild stains/isolates”. However, in a given variety, the mild stain status was maintained. We are evaluating the factors that affect the symptoms and titers and determining if a mild strain can be maintained in major commercial citrus varieties. Due to the slow hiring process of ARS, we have made a limited progress in this reporting period of time.
The objectives of this research are 1) to develop cost effective thermotherapy protocols for field application by optimizing temperature and relative humidity conditions in the tent; 2) to develop a mathematical model derived from our data and grower’s data which will be used to determine the best treatment duration in future applications; and 3) to study gene expression of HLB-affected citrus plants that received heat treatment, and identify critical citrus genes that may be induced by heat stress for the benefit of suppressing HLB. We are continuing the field samples and data analysis on heat-treated HLB-affected trees. We are in the process of determining an algorithm that relates environmental conditions with decreases in Las titer. In term of transcriptome analyses, we have conducted a comparison study between heat-treated and non-heat-treated citrus plants. There were 31 consistent up-regulated genes and 47 down-regulated genes in the the citrus trees treated with heating. We also conducted heat-treatment with constant temperature at 40’C, 85 % relative humidity, and a 12 hr photoperiod for 4 days. RNA samples were prepared with this group of trees and control trees, and sent for RNA-sequencing. Currently, we are analyzing these RNA-seq data, and we are verifying the putative important genes with differential expression(DE) by RT-PCR.
Stress intolerance of HLB trees is a direct consequence of greater than 30 percent loss of fibrous root density compared to non-diseased trees. This root loss may be compounded an additional 20% by the interaction with bicarbonates in the rhizosphere as a result of irrigation with well water high in bicarbonates and/or over-liming with dolomite to avoid copper toxicity. HLB and bicarbonate stress also favor infection by root pathogens such as Phytophthora spp. In field surveys and greenhouse seedlings of different rootstocks with and without Liberibacter asiaticus (Las) inoculation, populations of Phytophthora spp. are often elevated in the rhizosphere of HLB infected plants. When Las interacts with Phytophthora, infection of fibrous roots may be temporarily greater than that caused by Las alone depending on the stage of Las infection and root damage. Las infection disrupts of root function by altering the concentration of soluble sugars and increasing root exudation that attracts and accelerates infection by P. nicotianae. This exacerbation continues until the Las causes root death at which point populations of P. nicotianae rapidly decline. Similar increases and decreases of Phytophthora populations have been observed in the statewide Phytophthora survey conducted Syngenta. Comparing rootstocks in the greenhouse, tolerance to Phytophthora is broken by the interaction with Las such that resistance to biotic or abiotic stress is greatly reduced which results in the premature root loss. This explains the reduction in root system capacity for water and nutrient uptake in field trees affected by HLB on all rootstocks. Moreover, normal annual cycles of fibrous root production appear to be disrupted on HLB affected trees. To confirm this, bimonthly changes in fibrous root mass density and rhizosphere populations of Phytophthora are currently being surveyed in 8 ridge and 4 flatwoods sites in south central Florida.
Installation and sampling with root cages to quantify seasonal changes in root density continues in two field sites. In the first site (Hamlins), 6 months of root growth and root density in healthy and infected trees has been assessed. Sampling revealed seasonal variation in root infection and apparent shifts in the root flush cycle caused by Liberibacter asiaticus (Las). The initial sampling of root cages at the second field site (Valencia) was completed. Installation of root cages in a second site was necessitated and delayed by efforts to find new healthy trees as the previously sampled trees became positive. The development of the Valencia site provides additional insight into how root growth dynamics differ between early season Hamlins and late season Valencias based on different seasonal carbohydrate allocation for fruit ripening. We are also investigating non-destructive ways to measure root growth in the field that will allow for more precise determination of fibrous root longevity. As more healthy trees become PCR positive for Las it is increasingly difficult to find sufficient putative healthy trees in the field. It is especially hard to find trees of more advanced age for seasonal root sampling. As field sampling proceeds, it will become more of a description of HLB-affected tree decline than a comparison to healthy trees. Therefore, emphasis will continue to shift to greenhouse experiments for valid healthy vs HLB comparisons. Sampling at a rootstock trial site is underway with a year and a half of data on the effects of HLB on new experimental rootstocks. This sampling has begun to reveal how these new rootstock lines respond to Las infection. Data for these rootstocks is currently being summarized to present results at upcoming grower meetings. The two most promising rootstock lines and Swingle as a standard rootstock control have been graft inoculated in the greenhouse and transplanted into rhizotrons to monitor root growth and death. Two months after transplant, Las was detected at similar incidence in roots (up to 50% of inoculated trees) for all rootstocks tested. Currently root growth and root longevity in response to Las infection is being assessed.
FireWall 50WP (65.8% streptomycin sulfate; Agrosource, Inc.) has been granted a second year of 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 of FireWall registration, EPA requires monitoring of Xanthomonas citri subsp. citri (Xcc) for streptomycin resistance and cross resistance to human antibiotics in treated groves. The objective of this survey is to comply with EPA’s protocol for evaluation of streptomycin resistance for Xcc and risk of cross resistance for other leaf and soil bacteria in the treated locations. This protocol is based on one developed by Dr. George Sundin at Michigan State Univ. for the section 18 use of the antibiotic kasugamycin against the Fire blight pathogen. I will be meeting with Dr. Sundin at the annual meeting of the American Phytopathological Society in August to discuss application of his protocol for our survey.
Objective 1. To define the role of chemotaxis in the location and early attachment to the leaf and fruit surface. An improved assay to evaluate the chemotactic response of Xanthomonads to different compounds was tested. A paper disk impregnated with the test compound was placed on semisolid agar plate to establish a chemical gradient. A disk soaked with sterile distilled water was positioned on the opposite side of the agar plate with a drop of a bacterial suspension placed in the middle. After three days incubation, asymmetric colonies developed when the compound produced a positive chemotactic effect. Assays with Xcc 306 strain confirmed a positive chemotactic response for serine and apoplastic fluid from sweet orange leaves. This assay is currently being used to compare the effect of apoplastic fluids from sweet orange, Key lime, kumquat, grapefruit, lemon, cabbage and Prunus spp. for strains Xcc A, A*, Aw, X. alfalfae subsp. citrumelonis, X. campestris pv. campestris, and X. arboricola pv. pruni. Objective 2. To investigate biofilm formation and composition and its relationship with bacteria structures related with motility in different strains of Xcc and comparison to non-canker causing xanthomonads. Biofilm formation was evaluated for wide (Xcc 306, Xcc 62), and restricted (Xcc Aw, Xcc Iran2) host range strains of Xcc, X. alfalfae subsp. citrumelonis and X. campestris pv. campestris. Strains responded positively to apoplast fluids from sweet orange, Key lime, kumquat and cabbage on LB agar medium rich in nutrients or XVM2 media that models nutrient availability in the apoplast. Differences in biofilm formation were related to the nutrient availability of the culture media. In nutrient limited medium XVM2, apoplast fluid from sweet orange, Key lime and kumquat reduced biofilm formation for all the Xanthomonas strains evaluated. While in LB medium the response of Xcc strains was mostly an increase in biofilm formation. In contrast response of X. campestris pv. campestris was variable. Currently, the effect of DNAse treatments on surface movements on semisolid culture medium plates is being evaluated for all the strains mentioned above.
In the 6 year old block of Valencia on Swingle in Lake Placid, PCR positive trees with symptoms or pre-symptomatic were were treated with soil drenches of Magna-Bon (MB), Cop-R-Quik (CQ) and an experimental compound (EXP) with well demonstrated systemic activity against citrus canker (caused by Xanthomanas citri subsp. citri) as a soil drench. After two seasons of spring and fall soil drench applications with high and low rates of each of these compounds in 3 replicated blocks, visual tree health ratings on a scale of 1-5 (1=Vigorous, asymptomatic, 2=Slight decline; 3=Moderate decline; 4=Severe decline; 5=Non viable, won’t recover) were higher for treated trees than the untreated check. Tree responses indicate that these treatment were having phytotoxic effects on HLB infected trees rather than achieving reduction in bacterial infection and or reduction in HLB symptom expression.
The purpose of this project was to collect additional data from growers so that the efficacy of the enhanced nutrient programs could be assessed more fully after some promising results were obtained from the limited data collected last year. After extensive effort, some data were collected from three growers. However, none of the data sets was complete enough for analysis, and efforts to obtain the additional information failed. Consequently, we will not be able to complete the goals of this project. Because all involved (growers and researchers) were optimistic at the start of the study, some funds were spent so that graduate students would be prepared to conduct the analyses. However, when it became evident that data would not be forthcoming, expenditures stopped. We believe that the growers had full intentions of supplying the data. However, recent events, such as extensive premature fruit drop, have been devastating to the industry, and it is understandable that the growers no longer have the time or energy to help with this project.
Overall goal: To engineer Liberibacter asiaticus gene regulation in Sinorhizobium meliloti. Premise behind the work done thus far: ‘ We want test one Liberibacter asiaticus regulator as a pilot experiment, and will troubleshoot and make necessary adjustments before working with the other La regulators on our list. ‘ The sigma factor La.rpoH was chosen as a pilot experiment, given extensive data on the S. meliloti rpoH1 homolog, which can serve as a control. ‘ With synthetic La rpoH and Sm rpoH1 constructs, activity will be assessed on fusions to known S. meliloti RpoH1 targets. Goals: optimize induction time and IPTG (inducer) concentrations and measure reporter fusion activity. Conditions will be used for later global transcription analysis. Cloning synthetic allele of the Liberibacter asiaticus rpoH gene: ‘ A synthetic La rpoH gene was designed with optimized codon usage and ribosome binding site for expression in Sinorhizobium meliloti ‘ The complete DNA for La rpoH was synthesized using Invitrogen’s GeneArt gene synthesis. ‘ The synthetic DNA was cloned into expression plasmid, pSRK-Tc. This plasmid has an inducible lac promoter with low basal activity (Khan 2008, AEM 74:5053). The Tc plasmid succeeded as cloning vector, but failed to function in S. meliloti (see below) and we subsequently turned to two other versions with alternate resistance markers (Km, Gm). Cloning S. meliloti rpoH1 gene: ‘ rpoH1 was amplified from our rpoH1 clone using primers that introduced the same ribosome binding site and restriction sites as the La rpoH clone. ‘ After sequencing to confirm correct sequence, the rpoH1 gene was cloned into pSRK-Tc ‘ After vector failure, a second cloning placed the DNA into pSRK-Km and pSRK-Gm Conjugation of pSRK constructs into S. meliloti strains CL150 and RFF157 (.rpoH1): ‘ The initial pSRK-Tc constructs did not work, nor did the empty vector. Despite published reports of pSRK-Tc being used successfully in S. meliloti, in our hands the trans-conjugants do not grow to singles on M9 sucrose with Tc-10 or Tc-5. A positive control (rpoH1 in vector pRK290) was done at the same time and this yielded many Tcr transconjugants. ‘ The clonings were repeated with the two other versions of the plasmid (Gmr and Kmr). These constructs do appear to work in S. meliloti; they now must be confirmed by PCR, etc. Summary of accomplishments: A Liberibacter asiaticus gene, encoding the RNA polymerase sigma factor RpoH, was engineered for optimum expression in S. meliloti, synthesized, and successfully cloned into three different expression vectors. Conjugation of pSRK-Tc into S. meliloti did not work, due to apparent problems with the vector; conjugations with two alternate constructs were initiated and each gave promising results with abundant transconjugants.
Overall goal: To engineer Liberibacter asiaticus gene regulation in Sinorhizobium meliloti. Previous results: As a pilot experiment for study of Liberibacter asiaticus regulators, we synthesized the sigma factor La.rpoH using codons tailored for opimum expression in Sinorhizobium meliloti. We cloned this, and the comparable S. meliloti homolog, into Gm-resistant and Km resistant variants of vector pSRK. These vectors have an inducible lac promoter for varying the expression level of the sigma factor. These cloned genes and the empty vector control plasmid were conjugated into S. meliloti. New results: ‘ To assess function, we tested these constructs in RFF157 (.rpoH1) compared to wild type CL150, to see if they allow growth on LB at 37 degrees. Specifically it is known previously that WT grows at 37, but RFF157 cannot grow at the higher (restrictive) temperature o The La rpoH and Sm rpoH1 constructs allow growth at 37 degrees. pSRK empty vectors do not. From this, we conclude that the Liberibacter RpoH (La RpoH) protein is successfully expressed from pSRK, and functions in S. meliloti to replace the endogenous Sm RpoH. o We asked whether expression from pSRK is tightly dependent on lac induction. We observed that La rpoH and Sm rpoH1 strains grow better on media with IPTG (inducing the lac promoter), but difference compared to the non-induced control is not large. This suggests that lac promoter may have leaky activity, and that the sigma factor clones are being expressed to some extent even without inducer. This evidence for likely background noise has implications for our eventual experiments in which we assay transcription profiles. o We also tested plasmids expressing La rpoH in RFF231 (mutated with deletions of both rpoH1 and rpoH2). The cloned La rpoH does allow growth at 37 degrees in the double mutant. ‘ The next step of functional assay is to test expression of specific genes. We have chosen to make beta-glucuronidase (uidA) and fluorescent protein (FP) gene fusions to endogenous S. meliloti genes with responsiveness to Sm RpoH. Conjugation of pSRK constructs into S. meliloti strains CL150 and RFF157 (.rpoH1): o This will provide a quantitative way to test activity of constructs ‘ 0.5 mM IPTG o We have chosen to construct fusions to five different promoters: trxB, groES5, hslV, clpB, ibpA o To date, we have both uidA and mCherry (a red FP) fusions, as single crossover of constructs into CL150 genome. o We have now conjugated La rpoH and Sm rpoH1 constructs, plus the plasmid with no cloned insert (empty vector control) into each of the fusion strains Summary of accomplishments: we showed that the Ca. Liberibacter asiaticus rpoH sigma factor gene can complement a growth defect in a S. meliloti rpoH mutant. We chose 5 genes whose promoters are likely to respond to RpoH protein, and we have introduced our newly cloned Liberibacter gene into strains bearing fusions to those promoters. Our next step is on assay gene expression effects of La RpoH.
Fruit harvests have now been completed for this study (comprising 2 seasons: 2012-2013 and 2013-2014, two harvest Hamlin and two Valencia/season), but sensory, biochemical and chemical analyses on frozen samples continue. All analyses have been completed for the first year (2012-2013 season) including trained and consumer sensory panels, chemical analyses of sugars, acids, aroma volatiles, vitamin C, limonoids, flavonoids, electronic tongue and of bacterial titer of the juice using qPCR. Sugar, acid and aroma volatile analyses have now been completed for the 2013-2014 season, but peel oil, limonoid and flavonoid as well as sensory analysis is still under way. Liberibacter titer in the juice was determined by qPCR for the Hamlin harvests but not Valencia as yet. So far physical fruit measurements show that fruit from HLB-infected trees for last season are smaller and greener than fruit from healthy trees, regardless of nutritional treatments of which 3 were investigated so far for the earlier harvests. Sugars (especially sucrose), acids and ratio were generally lower in HLB juice, and acids were sometimes higher in juice from symptomatic fruit, regardless of treatment, except for one nutritional treatment for Valencia in March, 2013. Bitter limonoids along with many other flavonoids were higher in HLB juice for the first season, regardless of nutritional treatment and especially in symptomatic fruit or fruit from severely infected trees, although this is more the case in early season harvests. So far, the nutritional treatments have not shown a consistent effect, but there are sporadic positive effects on flavor chemicals. The nutritional treatments did not show an effect on reduction of Liberbacter titer in the juice as evidenced by qPCR analysis so far for the first season, nor in the Hamlin harvests from the 2013-2014 season. Several times, however one nutritional treatment has shown the ability to make the juice taste sweeter although no major effect on sugars was found. Now for the second season, the same nutritional treatment has shown little to no reduction of sugars due to HLB for the two Hamlin harvests (12/13 and 1/14) and Valencia (5/14) while all other treatments showed sugar reduction due to HLB. One of the new nutritional treatments added for the Valencia 5/14 harvest also showed less reduction of sugars due to HLB. The electronic tongue (etongue) and nose could discriminate between juices from healthy, asymptomatic-HLB and symptomatic-HLB fruit, with the etongue being much more effective. The etongue could also discriminate the different nutritional treatments within a harvest, but was confounded by seasonal changes across harvests. The etongue was more effective for Hamlin than Valencia, reflecting the more severe HLB-induced flavor effects for Hamlin in the first season samples, but the spring, 2013, Valencia samples did show separation indicating that the disease is becoming more severe for Valencia. Etongue has not yet been run for the 2013-2014 season samples. Trained panel showed differences in perception of orange and grapefruit, fruity, green and stale flavors and sweet, sour, bitter, metallic, tingling, astingent and umami (salty) tastes. Consumer difference-from-control panels showed that the panelists detect differences that correlate to lower ratio and/or higher limonoids in HLB compared to healthy juice. The differences is greatest when there is both low ratio and high limonoids. Aroma volatile analysis, now completed through 2014 show that some tope notes, especially esters, are generally lower in HLB juice. Samples were taken in recent harvests for analysis of peel oil and were processed and now run on GC-O (GC olfectometry) and GS-MS, but data not yet tabulated and limonene was analyzed with not differences noted.
Putative transgenic plants of PP-2 hairpins (for suppression of PP-2 through RNAi to test possible reduction in vascular blockage even when CLas is present) and of PP-2 directly are grafted in the greenhouse and growing for transgene verification, replication and testing. 40 putative transgenic plants transformed with citGRP1 were tested by PCR and twenty two of them were confirmed with citGRP1 insertion. RNA was isolated from some and RT-PCR showed gene expression. Some transgenics with over-expression of citGRP1 increased resistance to canker by detached leaf assay and infiltration with Xanthomonas. About 10 transgenic Hamlin shoots with citGRP2 were rooted in the medium and nine of them were planted in soil. Over 60 transgenic Carrizo with GRP2 were transferred to soil. DNA was isolated from 20 of them and 19 of them are PCR positive. Some of them showed canker resistance when infiltrated with Xcc at concentration of 105/CFU. Belknap reports that potatoes transformed with citGRP2 are displaying considerable resistance to Zebra Chip in Washington state. Fifteen transgenic Carrizo and seven transgenic Hamlin with peach dormancy related gene MADS6 were planted in soil and they are ready for DNA isolation. A chimeral construct that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab) is being tested. Many transformed Carrizo with the chimera AMP was obtained. DNA was isolated from 32 of them and PCR test confirmed 28 are positive. Canker test showed two of them greatly increased resistance at the infiltrated concentration of 107CFU/ml. DNA was isolated from 10 chimera transgenic Hamlin and PCR test confirmed 9 of them are positive. They will soon ready for RT-PCR for gene expression. Several transgenic Carrizo with thionin increased canker resistance remarkably with infiltration test at the concentration 107CFU/ml. RNA was isolated from transgenic plants containing chimera and thionin. RT-PCR showed gene expression in the transgenic plants. Further gene expression level was evaluated with RT-qPCR. Our results showed gene expression variation between different transgenic lines, from several fold to 35 fold. Transgenic lines containing D4E1 were evaluated with Xcc infiltration. All the transgenic lines with canker development at 105 CFU/ml while some transgenic lines show less canker development at 104 CFU/ml. Bacterial growth rate in transgenic lines containing D4E1, chimera and thionin was investigated by qPCR. Our results showed some transgenic lines containing chimera and thionin had low Xcc growth rate. To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was cloned into pBinARSplus vector (collaboration with Duan lab). Flagellins are frequently PAMPS (pathogenesis associated molecular patterns) in disease systems and CLas has a full flagellin gene despite having no flagella detected to date. The consensus FLS2 clone was obtained and used to transform Hamlin and Carrizo so that resistance transduction may be enhanced in citrus responding to HLB and other diseases. Many putative transformants were generated on the selective media. About ninety transgenic shoots were rooted with eighty Carrizo and ten Hamlin transformants planted in soil. DNA was isolated from 80 of them: 38 Carrizo and 7 Hamlin are positive by PCR test. Reactive Oxygen Species (ROS) assay showed typical ROS reaction in three of transgenic Hamlin which suggest nbFLS is functional in citrus PAMP-triggered immunity. However, there is only slight canker resistance by infiltration test. A series of transgenics scions produced in the last several years continue to move forward in the testing pipeline. Several D35S::D4E1 sweet oranges show initial growth in the field which exceeds that of controls. A large number of ubiquitin::D4E1 and WDV::D4E1 plants and smaller numbers with other AMPs are replicated and in early stages of testing.
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