In earlier reports, we have mentioned about superior antibacterial properties and strong retention of copper loaded silica nanogel (CuSiNG) material to plant surface, leading to improved efficacy against citrus canker disease. The objectives are to use less Cu and cut down number of spray applications without compromising protection against the citrus canker disease. To further improve the efficacy of CuSiNG material, we report here the outcome of our recent study on CuSiNG composite material and its antibacterial properties. The CuSiNG composite material has a core-shell nanoparticle design. The core is a pure silica nanoparticle core and the shell is made of CuSiNG material. The purpose of this design is to uniformly spread the active CuSiNG material (the shell layer) on to an inactive pure silica nanoparticle (the core) to take advantage of the high surface area of the nanoparticle core. Our 2011 field trial involves one spray application of the CuSiNG material in every three weeks. At this spray rate, it is expected that a significant amount of Cu in the CuSiNG formulations will remain unused or buried in the film/deposits. In other words, the unused portion of Cu could be potentially cut down from the original formulation without loss of antibacterial efficacy. To test the feasibility of this concept, the core-shell CuSiNG composite materials have been designed and studied. Particle size and the morphology of the core-shell CuSiNG composite material was characterized using transmission and scanning electron microscopy. The thickness of the CuSiNG shell was estimated to be ~ 50 nm. The silica nanoparticle core size was approximately 380 nm. Minimum inhibitory concentration (MIC) of the core-shell CuSiNG composite material was estimated to be 4.9 parts per million (ppm) Cu concentration against both E.coli and B.subtilis. In comparison to Kocide’ 3000 and copper sulfate containing equivalent amount of copper, the core-shell CuSiNG composite material exhibited a 50% and 70% decrease in bacterial growth, respectively. This preliminary study demonstrates that the efficacy of the CuSiNG material could be further improved by having a core-shell design. Note that the size of the silica nanoparticle core and the thickness of the CuSiNG shell are adjustable. This is particularly important as smaller size particles are expected to exhibit high surface area and strong retention properties. In coming reports, we will summarize the outcome of further studies on core-shell CuSiNG composite material. We will also report on the on-going fluorescence imaging studies to understand the interaction of CuSiNG material with bacteria as well as 2011 trial results on CuSiNG material.
In previous reports we have described the preparation of a scFv library prepared in phagemid vector pKM19. The basic scFv library contains 2 x 10_7th unique phage that bind to different antigens present in ‘Ca. Liberibacter asiaticus’ and the psyllid vector. We have also reported that we have isolated scFv from this library that bind to epitopes contained in proteins of ‘Ca. Liberibacter asiaticus’ that are likely to be related to host pathogen interactions and virulence. These epitopes are found on two flagellar proteins, the major outer membrane protein, a pilus protein, a protein believed to polymerize the capsular polysaccharide surface layer of the bacterium, the TolC protein required for survival in a plant host, and InvA, the invasiveness protein that prevents an infected cell from undergoing programmed cell death by apoptosis. The vector and expression system that we have used for this project allows selection of the scFv antibody expressed from a phagemid genome but packaged on the surface of an M13 particle. This facilitates selection, but large scale expression of the scFv requires cloning of the scFv gene into a cognate plasmid expression vector. We have had problems with many of our scFv at this step, because ‘stop’ codons can accumulate in the phagemid without affecting the selection process, but prevent expression from the plasmid vector. During this reporting period we have repaired the improper ‘stop’ codons for several of our scFv to allow full expression of the scFv. These scFv were selected because they showed the desired specificity when selected in the phagemid format, but failed to produce scFv when used in the expression vector. The repairs were done by sequencing the defective scFv and identifying the stop codons, designing primers for a series of PCR that allowed amplification of the scFv while replacing the incorrect codons with corrected sequence, and transforming the corrected plasmid with the scFv into E. coli for expression. We have corrected the sequences for scFv that bind FlhA (scFv B947, B1096, B1072); KpsA (B520, B1199, B1202); the major outer membrane protein OmpA (B743); Pilus protein (B556, B557). These scFv are now available in pKM16, our expression vector for testing. The next steps will require expression of the scFv, purification of the scFv and testing it for yield and specificity against purified antigens. These scFv will be added to our inventory of multiple scFv for each target of interest to improve our chances of finding scFv that will be extremely useful for detection assays and for labeling cells for scientific studies.
We continued working on the identification of sequencing variation within virulence-related genes and development of gene-based phenotyping markers for ‘Candidatus Liberibacter asiaticus’ (CLas). Previously, we selected four groups of virulent genes based on their putative functions in CLas genome. Sequencing analysis found that there were 1-3 single nucleotide polymorphisms (SNP) per gene locus when sequences were compared among HLB strains collected from US Florida, Brazil, China and India. BLASTx analysis indicated that there were two types of nucleotide variations; synonymous and non-synonymous variations. The former is defined as a silent substitution as such changes in nucleotide sequences will not alternate amino acid sequences due to the degeneracy of amino acid codons. Non-synonymous mutations, on the other hand, will result in change in amino acid sequences, thus would likely affect the function of the genes. Bacterial evolution and selection by specific host environments leading up to adaptation and speciation are a cumulative effect of gene loss and gain events. Sequence divergence in coding regions due to mutation accumulation in the individual genes could affect evolutionary fitness and pathogenic characteristics. To extend research, more virulence-related genes were investigated which included; Group 1, heat shock-related protein. In this group, DnaJ and DnaK chaperone proteins were cloned and sequenced. It was reported that the DnaK/DnaJ chaperone machinery was required in many bacteria that caused a systemic infection in the host. In Group 2, CLas secretion systems for type I signal peptidase, tolQ protein, integral membrane protein TerC, colicinV production protein and multidrug resistance transporter protein were cloned and sequenced. Genes involved in the transcriptional regulation system were also investigated (Group3). In this group, putative transcription regulator protein and LysR-type transcriptional regulator (LTTR) were selected. LTTR is one of the key players that help bacteria adapt to different environments. A hrg knockout mutant was found to be sensitive to oxidative products of the respiratory burst, specifically to hydrogen peroxide. A strain overexpressing the hrg gene showed a survival advantage over the wild-type Salmonella under hydrogen peroxide-induced stress. Group 4 contains pili and flagellar genes. Pili are essential for host colonization, virulence and pathogenesis for many bacteria. Flagella-dependent motility is widespread throughout prokaryotes and is advantageous when nutrients are limited. Flagella systems can also play an important role in additional processes such as adhesion to substrates, biofilm formation and host invasion in pathogenic bacteria. Seven CLas genes were identified in this group. They are: chemotaxis protein, pilus assembly protein, pilus assembly protein, flagellar basal body L-ring protein, flagellar biosynthesis regulatory protein FlaF and flagellar motor switch protein G, respectively. Finally, outer membrane protein OmpA and MotB were also included in this study. OmpA/MotB or OmpA is required for pathogenesis in many pathogenic bacteria, and can interact with host receptor molecules. MotB and or MotA serves two functions in E. coli; the MotA(4)-MotB(2) complex attaches to the cell wall via MotB to form the stator of the flagellar motor, and the MotA-MotB complex couples the flow of ions across the cell membrane to movement of the rotor. PCR-based gene cloning and sequencing for above selected gene loci are currently in process. We will establish an in vitro heterogous gene expression system to determinate and characterize the functions of these virulence-related genes.
To demonstrate that Candidatus Liberibacter bacterium is the causative agent of Huanglongbing disease (HLB), or greening, Koch’s postulates need to be performed with a pure culture of Liberibacter. We obtained primo-cultures of Ca. Liberibacter asiaticus (LAS) in insect cell cultures used as feeder cells and we improved our medium and growth conditions. From these primo-cultures we selected LAS cultures cured from insect cells and we checked whether LAS was the only bacteria in our cultures with broad-range PCR based on bacterial 16S rDNA. Other bacteria were found in co-culture with LAS (i.e. a Delftia acidovorans or an actinobacteria strain). Enforced vancomycin selection has been applied to get rid of gram positive contaminants, however LAS signal was lost along the selection. New antibiotics to select for LAS are under investigation. We set up a protocol for mechanical inoculation of the LAS culture to healthy citrus. Three independent LAS cultures (several transfers old) were inoculated to healthy citrus (2 young plants for each CIV). After 5 weeks of inoculation, the 6 inoculated plants were tested by PCR (on 5 leaves). Two plants, inoculated with two different bacterial cultures gave positive PCR detection of LAS. No HLB symptom has been observed to this day (9 weeks after inoculation). We will keep following the evolution of LAS signal in the inoculated trees and check for the onset of symptoms. Inoculations with different LAS CIV will be repeated. Another way to inoculate the LAS culture would be by acquisition through membrane by the insect vector and inoculation of healthy citrus via the infected insect. Experiments of acquisition through membranes are under investigation: psyllids starvation phase followed by a feeding phase of psyllids with the bacteria solution and finally a psyllid/citrus contact phase. Hopefully we will be able to inoculate soon a LAS pure culture to healthy citrus after elimination of contaminant bacteria.
To demonstrate that Candidatus Liberibacter bacterium is the causative agent of Huanglongbing disease (HLB), or greening, Koch’s postulates need to be performed with a pure culture of Liberibacter. We obtained primo-cultures of Ca. Liberibacter asiaticus (LAS) in insect cell cultures used as feeder cells and we improved our medium and growth conditions. From these primo-cultures we selected LAS cultures cured from insect cells and we checked whether LAS was the only bacteria in our cultures with broad-range PCR based on bacterial 16S rDNA. Other bacteria were found in co-culture with LAS (i.e. a Delftia acidovorans or an actinobacteria strain). Enforced vancomycin selection has been applied to get rid of gram positive contaminants, however LAS signal was lost along the selection. New antibiotics to select for LAS are under investigation. We set up a protocol for mechanical inoculation of the LAS culture to healthy citrus. Three independent LAS cultures (several transfers old) were inoculated to healthy citrus (2 young plants for each CIV). After 5 weeks of inoculation, the 6 inoculated plants were tested by PCR (on 5 leaves). Two plants, inoculated with two different bacterial cultures gave positive PCR detection of LAS. No HLB symptom has been observed to this day (9 weeks after inoculation). We will keep following the evolution of LAS signal in the inoculated trees and check for the onset of symptoms. Inoculations with different LAS CIV will be repeated. Another way to inoculate the LAS culture would be by acquisition through membrane by the insect vector and inoculation of healthy citrus via the infected insect. Experiments of acquisition through membranes are under investigation: psyllids starvation phase followed by a feeding phase of psyllids with the bacteria solution and finally a psyllid/citrus contact phase. Hopefully we will be able to inoculate soon a LAS pure culture to healthy citrus after elimination of contaminant bacteria.
This project is a continuation of a previous project #95 “PREPARATION OF ANTIBODIES AGAINST CANDIDATUS LIBERIBACTER ASIATICUS”. Progress reports for the previous project are on file. The reimbursable agreement with CRDF was established on September 5, 2012. Efforts have been underway during this quarter to recruit a visiting scientist to work on this project. No suitable candidate has yet been identified. We continue to study the literature to identify vectors to use for a future scFv library made as part of this project. The goal is to find a suitable vector that is not encumbered by intellectual property and patent issues. We are also optimizing the cloning strategies that will be used to move already selected scFv into transgenic plants.
This project is a continuation of a previous project #95 “PREPARATION OF ANTIBODIES AGAINST CANDIDATUS LIBERIBACTER ASIATICUS”. Progress reports for the previous project are on file. Permission to use the outside funds was received on September 5. A visiting scientist was hired and started work in October. A very well qualified candidate for the half time student aide job was identified but the hiring process has not yet been completed. Cultures of the clones expressing scFv were revived from storage. Initial efforts have been to improve the yield of scFv expressed from plasmid pKM16 in E. coli. Yields of protein were less than what we had expected. Others working with scFv have had similar problems. Review of the literature provided options to move forward. We have changed the culture medium for the scFv expressing E. coli to a much richer medium and culture at 30C. Differing amounts of IPTG have been tested to better control the induction of expression of the scFv. Yields of scFv proteins have improved. The scFV have been only modestly successful in ELISA, with the absorbance of positive samples always greater than, but usually less than 2X healthy controls. Best results were with scFv to the outer membrane protein of ‘Ca. Liberibacter asiaticus’. Results from dot blots are better, but background absorbance from healthy plant tissue is occasionally a problem. We are currently working on using the scFv in tissue printing assays, because with a low magnification stereomicroscope we can visualize the stain in the phloem tissue to give a better result. During this quarter we have also obtained repairs on our fluorescent light microscope and will use it with fluorescently labeled scFv to detect the pathogen in plant samples.
This project is a continuation of a previous project #95 “PREPARATION OF ANTIBODIES AGAINST CANDIDATUS LIBERIBACTER ASIATICUS”. Progress reports for the previous project are on file. There is no research progress to report at this time on the current project. Funding has not been received.
This project is a continuation of a previous project #95 “PREPARATION OF ANTIBODIES AGAINST CANDIDATUS LIBERIBACTER ASIATICUS”. Progress reports for the previous project are on file. There is no research progress to report at this time on the current project. The contract was put in place during this quarter and I received permission to use external funds on September 5, 2012. Efforts to recruit a visiting scientist and student aide to perform the research work were made during the summer months. A visiting scientist was identified and the visa and other arrangements were made.
This project is a continuation of a previous project #95 “PREPARATION OF ANTIBODIES AGAINST CANDIDATUS LIBERIBACTER ASIATICUS”. Progress reports for the previous project are on file. There is no research progress to report at this time on the current project. Funding has not been received.
In this report we will show the final results obtained with serological and molecular approaches used for diagnosis of Liberibacter asiaticus. The molecular approach by Nested PCR (N-PCR) protocol was finalized and definitively the double step was much more sensitive than the one step (one single tube Nested PCR). We used DNA from a single psyllid or from inoculated citrus plants by psyllids as template for quantitative PCR (QPCR) and the results were compared against Nested PCR one single step (NPCR-OS), double step (NPCR-DS), and traditional PCR (T-PCR). We verified that a hundred percent of matches between QPCR and NPCR-DS was obtained for the samples whose threshold cycle (Ct) values ranged from 22 to 32, as determined by QPCR. For the samples whose Ct values ranged from 32.1 to 34, and from 34.1 to 36 there were 98.11% and 96.61% of matches between NPCR-DS and QPCR, respectively. For those samples with Ct values above 36.1 only 54% of results were shared between QPCR and NPCR-DS. The number of positive samples determined by NPCR-OS was lower than determined by NPCR-DS or QPCR, but a higher when compared to traditional PCR for all comparison levels. Finalizing, based on the significant agreement between NPCR-DS and QPCR for the samples with Ct <36 as well as others facts, as the non-frequently Ct around of 38 reported for the negative controls, we used the 36th cycle as a threshold for decide if the samples are positive (Ct <36) or negative (Ct >36). The NPCR-DS primers are based on Outer Membrane Protein (OMP) gene and the sequence follow; (OMP1F-tgtaattcggcgtgaacttg and OMP1R-cacgcggacctataccctta -884 pb fragment) and the internal primers (OMP2F-ggcgtagaagggcatattga and OMP2R-acgtggcacaattgggttat -411 pb) for the second round of PCR. For the serologic-based diagnosis we showed in the last report four antibodies (T3699-4, T3699-5, T3699-6, T3699-7) with potential to be used in ELISA for diagnosis of Las. Of those the antibody T3699-7 that recognize a fraction of outer membrane lipoprotein by the sequence ILSYFQKDGKFKTISTDGS (NCBI ID = YP_003064612.1,) shown the best results, including with positive reactions in Dot blot assay. After that, as proposed, we started to test this antibody on a broad number of HLB symptomatic samples. Inexplicably no more reactions against disease samples were observed with this antibody. After this antibody was purified using chromatography columns and again no more reactions were observed. We also did new antibodies but the negative results were persistently obtained. We really don’t know how to explain this lost of reactivity by the antibody T3699-7 and also by the others,T3699-4, T3699-5, and T3699-6 . Those were tested on several host species and different type of HLB symptoms. Finally, in meantime of T3699-7 test we were ask for send the antibody to Dr. Nabil Killiny (UF – Lake Alfred), Dra. Folimonova (UF – Lake Alfred), and Dr. Ammar Desouky (USDA – Fort Pierce) witch was done. Some success of use of the T3699-7 was reported for us by Dr. Killiny. Although the project was officially concluded on last 2011, March, we still are working on to figure out what could be happen with the T3699-7 antibody.
Thus far, 646 different media formulations have been tested to support growth of Candidatus Liberibacter asiaticus under various culture conditions. None of the media supported planktonic growth of the bacterium. Biofilm formation was observed on the surface of the alimentary canals of Diaphorina citri used as a source of inoculum. These biofilms were more extensive in some media than in others. The addition of mixtures of essential and of non-essential amino acids appeared to promote biofilm formation. Biofilm formation was more evident under aerobic and in microaerophilic conditions than under anaerobic conditions.
Discovery of causal agents is important to prevent disease dissemination. Pathogens evolve continuously and a large number of graft-transmissible citrus diseases indicate that viruses and viroids the most likely causative agents. In California, Citrus Clonal Protection Program (CCPP) offers quarantine disease testing and budwood certification services. Quality control using traditional immunological or nucleic acid based methods are great, but are limited to only known pathogens and diseases. Now, the major concern is to develop protocols to identify and catalog previously uncharacterized viruses and viroids to achieve the primary goal of CCPP to provide disease free citrus cuttings to the industry and farmers. My lab has been successfully developed a novel method known as vdSAR to identify viruses from insects and plants (Wu et al., 2010). This approach is based on the fact that virus-specific small interfering RNAs (viRNAs) produced in infected plant and insect cells are overlapping and can be assembled back into large fragment of the invading viral genomes. Thus, deep sequencing and assembly of total small RNAs from the disease samples of citrus will likely identify previously unknown viruses. Viroids are a distinct class of free RNA pathogens that cause diseases in citrus and other plant species. However, current approaches for viroid discovery are time-consuming and technically demanding so that only a small number of viroid species have been identified in the last 40 years. It is therefore likely that some of the unknown graft-transmissible citrus diseases are caused by viroids. Thus, we have focused on the development of a deep sequencing-based approach for viroid discovery in the last year. We found that viroid-specific siRNAs accumulated in infected plants are also overlapping and that full-length circular viroid genomic RNA can be assembled by a new computational algorithm we have developed (Wu et al., in preparation). We have tested this approach for viroid discovery using a published small RNA library made from Italian grapevine and discovered a new viroid that shares no significant homology to any known viroid. We have obtained nine graft-transmissible citrus disease samples from our collaborator Dr. Vidalakis laboratory, UCR. In the first batch, we have completed small RNA library construction for four samples: Yellow Vein and Vein Enation, Fukumoto decline on trifoliate and Beck. Using our approach, we predicted two candidate viroids (Vd-1 and Vd-2) from the yellow vein small RNA library. Based on the predicted sequence, we were able to amplify and clone Vd1 (276 nt) and Vd2 (234 nt) from Yellow Vein using RT-PCR and to show that Vd1 existed as a circular molecule. We are in the process of testing the infectious nature of Vd1 in plants. In the second batch, we have constructed small RNA libraries for five more samples: Concave Gum, Cristocortis and Fatal Yellows (CCPP, UCR), Bahia scale bark and Leprosis (nuclear type) from Brazil. Deep sequencing of small RNAs is in progress. Once the data is received, we will adapt our vdSAR strategy to discover viruses and viroids from these samples.
Detection of genetic diversity within pathogen populations is fundamental for ecological and epidemiological studies of a disease. The genetic structure within a given pathogen population is an indispensable prerequisite for determining sources of infection and risk management of the disease. The microsatellite markers developed in this project provided a useful tool for detection and genetic diversity study of ‘Ca. L. asiaticus’ isolates for global populations and the epidemiology of HLB. A panel of seven polymorphic microsatellite markers was developed. Microsatellite analyses amongst 134 Las isolates from Asian and American continents showed that the genetic diversity of ‘Ca. L. asiaticus’- is higher in Asia than diversity in the Americas. UPGMA and STRUCTURE analyses identified three major genetic lineages of ‘Ca. L. asiaticus’ worldwide. Isolates from India were most genetically distinct compared to other countries. East-southeast Asian and Brazilian isolates were generally included in the same group, few members of this group were identified in Florida, but the majority of the isolates from Florida were clustered in a separate group. Isolates from Brazil showed similar genetic makeup with east-southeast Asian dominated groups, suggesting the possibility of a common origin. In contrast, most of the isolates from Florida were clustered in a separate group. While the actual sources of the dominant ‘Ca. L. asiaticus’ strains in Florida are still in question, the genetic analysis from this study suggests that the introduction of less- pervasive strains may have been introduced directly from Asia or via Brazil based on their genetic similarity. However, the recent outbreak of HLB in Florida is predicted to have occurred from multiple introduction events. The multilocus microsatellite marker system developed in this study provides adequate discriminatory power for the identification and differentiation of closely-related ‘Ca. L. asiaticus’ strains, as well as for population genetic studies of the HLB-associated Liberibacters. Based on the reference genome of Florida isolates, we identified pathogenic genes that are located in or near these regions. The gene-based of new molecular markers were thus designed and classified into four major catalogues: 1) Phage-derived genes which are often involved in pathogenesis through their transfer of genes and/or genetic rearrangement. 2) Genes defined as two component response regulators. This group of genes is important for sensing environmental signals and producing a transcriptional response from those signals. 3) Genes identified with hydrolase activity. One of the interesting genes in this group is salicylic acid hydrolase which functions as hydrolysis of salicylic acid, a signaling molecule expressed in host associated with defense response to pathogen attack. However, to confirm its hydrolase function, the substrate for this gene-coded enzyme needs to be determined. 4) Zinc binding proteins, znuABC genes identified as putative high-affinity zinc uptake system that belongs to the ATP-binding component of ABC transporter protein. Zn uptake system, znuABC is required for obligate bacterial zinc homeostasis in intracellular environments which contributes to the virulence of Las in citrus plants. To provide insight of genetic variation of Las at whole genome level, more recently, we successfully obtained the whole genome sequence of a Chinese Las strain using Illumina sequencing technology. Preliminary genome analyses revealed that sequence variations were observed between Florida and Chinese strains. Research is in process toward the completion of sequence contig validation and connection. Comparative genomic analyses will provide critical information about the evolutionary history of genetic relationships between Asian and American strains. The availability of additional HLB-associated Liberibacter genomes will also facilitate development of gene-based markers that can be used for strain characterization.
We have made substantial progress in the first two objectives of the proposal and the citrus plants containing some of HLB effectors are being challenged inoculated with HLB through psyllids which is the third objective. Based on the sequence information number of putative effectors unique to Candidatus Liberibacter asiaticus, the causal agent of citrus Huanglongbing (HLB), present in prophage related gene clusters and/or pathogenicity islands have been identified and are believed to contain bacterial pathogenicity, multiplication and virulence determinants. We have cloned most of the effectors into binary vector based citrus tristeza virus (CTV) vector, between CP and CP ORFs as a separate cistron under an heterologous beet yellows virus coat protein controller element and were agro-inoculated into leaves of Nicotiana benthamiana as outlined in earlier progress reports. Partially purified virions of recombinant CTV containing HLB effectors from the infiltrated leaves and /or the systemic leaves of N. benthamiana have been used to inoculate Citrus macrophylla plants. 15 individual HLB effector genes are in citrus ready for challenge inoculation with HLB and many more in N. benthamiana at different stages of transfer to citrus. Observations of Mike Irey of US Sugar have indicated that sweet orange is the preferred host for screening with HLB as against Citrus macrophylla. Thus, we will use sweet orange citrus cultivar for primary inoculations from N. benthamiana. We have also cloned some of the effectors into the 3′ end of CTV between p23 ORF and the 3′ non-translated region of CTV based on the recent finding of augmented expression of an inserted foreign gene. One of the important effectors or virulence factors is hemolysin of HLB is a 50 kDa protein secreted by type I secretion system. The hypothesis is that the hemolysin might interfere with metal ion transportation leading to host metabolic imbalance potentially resulting in disease symptoms. An abstract entitled ‘Functional studies of Hemolysin of ‘Candidatus Liberibacter asiaticus’ in citrus using citrus tristeza virus vector’ was presented at the 2011 American Phytopathological Society meeting and we plan to publish together with results of HLB challenge inoculation. Additionally, we are using a faster approach to screen for virulence genes of HLB using Potato virus x (PVX) vector system which has been used extensively to screen for virulence genes in herbaceous hosts. Agrobacterium-mediated transient expression of 34 genes derived from CLas prophage-related gene cluster into PVX vector, did not induce obvious phenotype changes in tobacco plant. However, transient expression of CLas-flagellin, flg22Las induced cell death and callose deposition in N. benthamiana, while the transcription of Bak1 and SGT1 associated with plant innate immunity were up-regulated. Substitution experiments revealed that the 38th and 39th amino acid residues were essential for callose induction. The synthetic flg22Las peptide (DRVSSGLRVSDAADNAAYWSIA) could not induce cell death, but retained the ability to induce callose deposition at the minimal concentration of 20 ‘M. These results demonstrated that Flg22Las has PAMP activity, and may play an important role in determining citrus resistance to CLas. These results were presented at the 2011 American Phytopathological Society meeting and a manuscript will be submitted for publication in Phytopatholgy.
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