Progress 07/01/09 to 09/30/13
Outputs
Target Audience:The targeted audience citrus growers, virologists, and plant pathologists were reached via formal and informal publications, and presentations in citrus grower meetings and at the Southwest Ag Summit meetings. The targeted audience of high school students and college undergraduate and graduate students were reached via classroom instructions and laboratory exercises. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Eight undergraduate students took part in the research activities during the 5-year project period. They learned not only basic molecular techniques, but also scientific approaches and problem solving skills. How have the results been disseminated to communities of interest?Most results from this project have been presented in professional journals, annual meetings of the American Phytopathological Society, and meetings and workshops of citrus growers and professionals. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
IMPACT of this project: Citrus tristeza virus (CTV), and the disease it causes, are an enduring threat to the U.S. and world-wide citrus industries. Substantial costs are involved in disease management. Hundreds, if not thousands of CTV isolates have already been described to have a wide range of host specificity and cause a variety of disease phenotypes. Most natural CTV infections were caused by a complex of CTV consisting of multiple strains. Within this complex, promiscuous recombination between CTV genomes occurred at remarkably high rates. The diversity of CTV and its interaction with the citrus hosts constitute a complex pathosystem. Some CTV genotypes prevent superinfection of other CTV genotypes (cross-protection), while others co-exist in complexes along with abundant recombinants; whereas sequences of a single, pure CTV genotype remain stable for long periods of time, new pathogenic isolates of CTV continue to emerge. Our results suggest that the high level of promiscuous RNA recombination in CTV compensates for the extreme genome stability and the low mutation rate, and functions as a major force driving the production of genetic variants important for adaptation and evolution. Effective management of CTV is achievable if we can attain a sound understanding of CTV genomic diversity, develop a rapid and accurate CTV identification tool, and gain an in-depth understanding of the mechanisms of CTV evolution. Outcomes of this project provided further understandings in these aspects, and made contributions to more effective CTV management. Accomplishments: Objective 1. CTV genomic analysis using resequencing microarray 1.1. To characterize multiple strains in natural isolates and aphid-transmitted progenies We designed a CTV resequencing microarray representing a genetic diversity equivalent nearly to ten full-length CTV genomes. The efficacy and accuracy of the resequencing microarray were validated using cloned T36 and T30 genomes, yielding, for the two reference target DNA preparations, call rates of above 99% and call accuracies of 99.9% to 100%. This performance is therefore comparable to that described for the SARS virus resequencing microarray, but better than that described for the Bacillus anthracis microarrays. We have successfully applied the CTV resequencing microarray to analysis of eleven natural CTV isolates from Florida, five aphid-transmitted CTV isolates from California, two defective CTV genomes from Spain, and one isolate from Arizona (unpublished data). Of the eleven Florida field isolates, eight contained two or more distinct CTV genotypes. Among the five aphid-transmitted California CTV isolates, four contained a single genotype while the other isolate contained a predominant genotype and a minor genotype, suggesting that aphids can select a single genotype from a viral complex but can also transmit multiple genotypes. 1.2. To determine genomic stability and mutation rates of CTV progenies from an infectious cDNA clone Using the CTV microarray, we demonstrated that CTV has extraordinary genome stability over a long period of time and has a mutation rate significantly lower than those of most RNA viruses. A genome-wide resequencing analysis of six progeny isolates derived from a T36 infectious cDNA clone, provided by Dr. Bill Dawson, showed only two to three fixed mutations in each genome. Since these isolates had been maintained in a variety of citrus species from one to over six years, these results suggested that the genomes of these pure CTV isolates had remained extraordinarily stable, regardless of the citrus species and the lengths of infection in the hosts. The mutation rates in these CTV progenies were calculated to be one to two magnitudes lower than those of other RNA viruses, and comparable to that of Hepatitis virus B, a pararetrovirus with a dsDNA genome. Objective 2. CTV recombination and host selection on CTV population dynamics 2.1.To characterize by ultra-deep 454 sequencing the global genotype composition of two natural CTV isolates, FS2-2 and FS2-8, and their progeny isolates transmitted to hosts with differential resistance to some strains CTV isolates, FS2-2 and FS2-8, are two sister isolates discovered several trees apart in the same citrus grove and are associated with an unusual stem-pitting symptom in the Hamlin sweet orange tree in Florida. Both contain a CTV complex consisting of three dominant strains and abundant recombinants. We used 454 GS-FLX to sequence a population of CTV genomes in the FS2-2 CTV complex. We first amplified CTV genomes as four RT-PCR fragments by long range RT-PCR using universal primers that are conserved in all known CTV genomes. Equal molar amounts of purified RT-PCR fragment were pooled together for 454 sequencing. Sequencing reads were assembled into three separate, full-length CTV genomic contigs, using the CodonCode Aligner assembler. The three contigs corresponded to the genome of CTV T30, T36 and VT genotypes. Further analysis of the NGS data confirmed the genome stability of CTV as the nucleotide diversity of the population of CTV genomes sequences was two to three magnitudes lower than that of other RNA viruses. Additionally, 5% of the 454 reads turned out to be recombinants between CTV T30, T36, and VT genotypes. 2.2.To identify and map recombinants from these CTV isolates and to confirm recombinants by direct sequencing of targeted regions Out of the more than 8200 sequence reads from our preliminary 454 sequencing of FS2-2, we identified 188 sequencing reads as recombinant sequences. The parental sequences for each recombinant were readily identified among the three major genotypes. The recombination crossover sites contained a stretch of sequence that was identical in both parental molecules. Nearly half recombinant reads were also assembled into contigs of mostly 2 sequence reads with a recombinant contig comprising 9 sequence reads and an assembled length of 1068 nucleotides. This recombinant contig is composed of mosaic sequences from all three genotypes joined together with 4 crossover sites (T36-VT-T36-VT-T30). When the recombinant sequence reads were mapped back to the genomes of the three strains in the FS2-2 complex, the resulting recombination maps revealed an astonishing level of recombination activities across the entire genome, with most active recombination occurring at the 3' half of the CTV genome. 2.3.To investigate the effects of selective citrus hosts on the fate of different CTV strains and recombinants. The CTV complexes in FS2-2 and FS2-8 had already been graft-transmitted to three different citrus species in 2004 in Dr. Dawson's laboratory: Valencia sweet orange (C. sinensis), Sun Chu Sha mandarin (SCS) (C. reticulata), and Swingle citrumelo (C. paradise x Poncirus trifoliata hybrid). These will be considered as daughter isolates. Swingle daughter isolates of both FS2-2 and FS2-8 origins were subsequently graft-transmitted to SCS in 2006 to produce the third generation isolates, designated as SCS/Swingle isolates. SCS and Swingle are commonly used as CTV-resistant rootstock. SCS is considered more resistant to T36-like genotypes while Swingle is considered more resistant to T30-like strains. Analysis of these progeny isolates confirmed the differential resistance to, or selection against certain CTV genotypes in these isolates. Passages through Swingle plants have the most remarkable effect on the T30 strain as it is virtually eliminated in FS2-2-Swingle. The effect of SCS on the T36 genotype is equally as dramatic, but a small amount of T36 remained detectable. And the VT genotype was not suppressed in all host plants. Suppressed strains appear to recover somewhat but not fully after being transferred to a series suppressed hosts. These data suggest that host resistance play important role in the CTV evolution.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Xiong, Z., Wong, B., Yu, N., Cant�a, J., Allee, E., Cochran, A., and Trinh, S. (2012). Biological and molecular characterization of Tomato chlorotic dwarf viroid in Arizona. Phytopathology 102, S139-S139
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Acosta-Leal, R., and Xiong, Z. (2013) Interference among co-infecting variants and fitness tradeoff condition the emergence of resistance breaking variants of Potato virus Y. Virology.
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Felix-Gastelum, R., Cochran, A., Yu, N., Herrera-Rodriguez, G., Trinh, T., Martinez-Valenzuela, C., and Xiong, Z. (2012). Genetic diversity and whitefly transmission of Tomato apex necrosis virus. Phytopathology 102, S37-S37
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
McRae, A. G. , Cochran, A, Trinh, S., Felix-Gastelum, R., Herrera-Rodriguez, G., Yu, N., and Xiong, Z. (2013) A torradovirus complex in Sinaloa, Mexico. Phytopathology 103, S94-S94
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Progress 01/01/12 to 09/30/12
Outputs
Target Audience: Citrus growers, virologists, and plant pathologists. These audiences were reached via formal and informal publications Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Two undergraduate students took part in the research activities under this project. They learned not only basic molecular techniques, but also scientific approaches and problem solving skills. How have the results been disseminated to communities of interest? Results from this set of experiments were presented at the International Organization of Citrus Virologists Conference in Argentine to citrus virologists from all over the world and at the workshop "Microarrays and Next-Generation Sequencing for Detection and Identification of Plant Viruses" sponsored by BARD at Beltsville, MD to a group of selected virologists from any countries. What do you plan to do during the next reporting period to accomplish the goals? Data generated in this period will be analyzed exhaustively; manuscripts describing these results will be prepared.
Impacts
What was accomplished under these goals?
To understand how CTV evolves under the selection pressure imposed by hosts, we analyzed CTV populations passed through a combination of two citrus varieties that may differentially suppress certain CTV strains: the Sun Chu Sha (SCS) mandarin and the Swingle citrumelo. The CTV isolate used for this passage experiments was FS2-2, a natural field isolate that contains a mixture of the T30, T36, and VT strains and has been maintained on the Valencia citrus. This parental isolate was graft-inoculated to SCS and Swingle to produce progeny isolates FS2-SCS and FS2-Swingle. Two independent FS2-Swingle isolates were subsequently graft-inoculated to SCS to obtain isolates FS2-SwingleA1/SCS and FS2-SwingleA3/SCS. These five isolates consisting of the parent, the first, and the second generation isolates were subjected to population analysis. Total RNA was extracted from the infected tissues of each of the five isolates and relative amounts of the three CTV strains in were measured by real-time reverse-transcription PCR, cloning and sequencing of selected cDNA genomic regions, and restriction fragment length analysis. Nearly 300 independent clones of CTV genomic fragments were analyzed in the process.
Publications
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: To understand how CTV evolves under the selection pressure imposed by hosts, we analyzed CTV populations passed through a combination of two citrus varieties that may differentially suppress certain CTV strains: the Sun Chu Sha (SCS) mandarin and the Swingle citrumelo. The CTV isolate used for this passage experiments was FS2-2, a natural field isolate that contains a mixture of the T30, T36, and VT strains and has been maintained on the Valencia citrus. This parental isolate was graft-inoculated to SCS and Swingle to produce progeny isolates FS2-SCS and FS2-Swingle. Two independent FS2-Swingle isolates were subsequently graft-inoculated to SCS to obtain isolates FS2-SwingleA1/SCS and FS2-SwingleA3/SCS. These five isolates consisting of the parent, the first, and the second generation isolates were subjected to population analysis. Total RNA was extracted from the infected tissues of each of the five isolates and relative amounts of the three CTV strains in were measured by real-time reverse-transcription PCR, cloning and sequencing of selected cDNA genomic regions, and restriction fragment length analysis. Nearly 300 independent clones of CTV genomic fragments were analyzed in the process. Results from this set of experiments were presented at the International Organization of Citrus Virologists Conference in Argentine to citrus virologists from all over the world and at the workshop "Microarrays and ext-Generation Sequencing for Detection and Identification of Plant Viruses" sponsored by BARD at Beltsville, MD to a group of selected virologists from any countries. PARTICIPANTS: Zhongguo Xiong (PI): design and execution of the overall project and experiments; Alicia Cochran, Bryant Wong, Eric Alle, and Sylvia Trinh (undergraduate students): participation in various experiments during the project; William Dawson (collaborator): provided various CTV isolates from Florida; David Galbraith (collaborator): experimental design and analysis. TARGET AUDIENCES: Citrus growers, virologists, and plant pathologists PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
In the CTV complex, we theorize that the interactions among strains and with their hosts drive the evolution of CTV. We already showed that promiscuous RNA recombination acted as a major force driving the genetic diversity and evolution of CTV. Results from the CTV passage experiments indicate that the hosts can also exert a transient but dramatic effect on CTV population dynamics and evolution. The parental Valencia isolate FS2-2 was found to contain mostly the VT strain with a low but readily detectable level of the T30 and T36 strains. When FS2-2 was passaged through the two hosts individually or sequentially, the VT strain remained as the predominant strain in all the progenies with its proportion ranging from 70% to nearly 90%. In FS2-SCS, the proportion of T36 was reduced dramatically to about 0.4% of the total viral population while the proportion of the T30 strain nearly tripled from 5% in Valencia to nearly 15% in SCS, indicating that SCS strongly selected against the T36 strain, and possibly promoted the T30 strain. Alternatively, the suppression of T36 left some empty biological spaces/territories that allowed the expansion of T30. In contrast, the passage of the parental isolate to Swingle nearly wiped out the T30 strain, which was barely detectable by the real-time PCR, suggesting that Swingle selects strongly against the T30 strain. Similarly as the T30 strains in SCS, the portion of the T36 strain increased to 12.5% in Swingle from 7% in Valencia. Interestingly, in the double passaged progenies, FS2-SwingleA1/SCS and FS2-SwingleA3/SCS, the T30 strain that was previously nearly gone in the parental FS2-Swingle isolates were now recovered with a thousand-fold increase. Interestingly, the amount of the T36 strain in the double passaged progenies was not suppressed by SCS as much as in the FS2-SCS isolate. These observations may be explained by a number of factors. First, the T36 strain is present at a larger proportion in Swingle (12.5%) than in Valencia citrus (7%), and is able to wither the suppression by SCS better. Secondly, the passage of CTV through Swingle may have acclimatized the T36 strain such that it can now withstand the suppression by SCS better. In general, both SCS and Swingle are less hospitable to CTV than Valencia, with SCS more so than Swingle. This is reflected in the total amount of CTV present in a unit of total RNA: 38.2 pg in Valencia, 17 pg in Swingle, 4.4 pg in SCS. It is worthy of mentioning that the absolute amount of CTV in SCS was slightly higher (5.6 and 8.6 pg) when the CTV isolate was acclimatized in Swingle first. These results indicated that SCS and Swingle could selectively suppressive but not completely eliminate the T36 and T30 strains, respectively, and the suppressed strains could recovered when transferred to a more permissive host. Other factors such as the overall virus load and the starting relative amount may also affect the suppressive effect of SCS on T36. The specific suppression of certain strains by a host creates a bottle neck that can drive the change in the evolutionary direction of the suppressed strains more so than in the absence of selection.
Publications
- Weng, Z., Liu, X., Yan, Z., Garnsey, S. M. Robertson, C. J., Dawson, W. O., and Xiong, Z. (2011). Differential suppression of Citrus tristeza virus strains by Sun Chu Sha mandarin and Swingle citrumelo. Proc. 18th Conf. IOCV (Campinas, Brazil, Nov 7-12. 2010)
- Weng, Z., Liu, X., Gowda, S., Barthelson, R. A., Galbraith, D. W., Dawson, W. O., and Xiong, Z. (2011). Extreme genome stability of Citrus tristeza virus. Proc. 18th Conf. IOCV (Campinas, Brazil, Nov 7-12. 2010)
- M. Saponari, A. Giampetruzzi, H. Doddapaneni, G.Loconsole, Z. Xiong, P. Saldarelli, R.K. Yokomi. 2011. Global analyses of small interfering RNAs from sour orange seedlings infected with different citrus tristeza virus genotypes. Proc. 18th Conf. IOCV (Campinas, Brazil, Nov 7-12. 2010
- Gong, D., Wang, J.-H., Lin, Z.-S., Zhang, S.-Y., Zhang, Y.-L., Yu, N.-T., Xiong, Z., and Liu, Z.-X. (2011) Genomic sequencing and analysis of Chilli ringspot virus, a novel potyvirus. Virus Gene 43:439-44.
- Xiong, Z., Weng, Z., Galbraith, D. W., and Dawson, W. O. (2010) Viral population analysis by genomic sequencing. Phytopathology 100:S154
- Xiong, Z and Acosta-Leal. R. (2011) Dissect the evolutionary process of Potato virus Y to overcome host resistance during single-host passages. Phytopathology 101:S194
- Acosta-Leal, R., Duffy, S., Xiong, Z., Hammond, R. W., and Elena, S. F. (2011) Advances in plant virus evolution: Translating evolutionary insights into better disease management. Phytopathology 101: 1136-1148. DOI: 10.1094/PHYTO-01-11-0017
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: CTV has one of the largest genomes among RNA viruses and consequently encodes more viral proteins with divergent functions and complex interactions. Its genetic diversity is one of the largest among RNA viruses. As a result of this genetic diversity, the virus has an extraordinarily large range of phenotypes, from no symptoms, to almost complete prevention of growth due to stem pitting, to rapid death due to graft incompatibility. This viral disease problem is further compounded by the fact that most natural CTV infections were caused by a CTV complex consisting of multiple strains. Within this complex, promiscuous recombination between CTV genomes occurred at remarkably high rates. This provides a unique opportunity to examine how the presence of mixed viral populations affects disease symptoms, and how different genotypes interact, co-evolve, and recombine over time, resulting in emerging new viral strains. In this project period, we applied the high-throughput 454 sequencing to sequencing a population of CTV genomes in a CTV complex fs2-2. The entire population of viral genomes in the complex was amplified by long range reverse transcription-polymerase chain reaction as four DNA fragments. Presence of three CTV strains in the FS2-2 complex was confirmed first by hybridization to the CTV resequencing microarray. Co-infection by T30-like (fs2_2_t30), T36-like (fs2_2_t36), and a VT-like (fs2_2_vt) CTV strains was evident. Sequencing by GS-FLX of the amplified CTV genomic fragments produced 8,722 high quality reads, having an average read length of 256 nucleotides, for a total of 2.23 megabases of CTV sequence. Most of the sequencing reads were assembled into three high quality, full-length genome contigs representing the genomes of fs2_2_t30, fs2_2_t36, and fs2_2_vt (Genbank accessions: EU937519-EU937521). The average coverage of the three contigs was between 27X and 42X. Examination of the assembled genome contigs revealed surprisingly few single nucleotide polymorphism (SNP) sites in the alignments of sequencing reads, despite the high coverage. The frequencies of SNP sites were 1.6 x 10-3 , 2.3 x 10-3 , and 1.0 x 10-3 for fs2_2_t30, fs2_2_t36, and fs2_2_vt, respectively. The nucleotide diversity () 17 was calculated as 3.97 x 10-4, 4.51 x 10-4, and 7.99 x 10-5 for the genomes of fs2_2_t30, fs2_2_t36, and fs2_2_vt, respectively. A substantial number of the 454 sequencing reads (203, 2.3% of total reads) were found to be recombinants comprised of sequences from two or more CTV strains that were joined together with no nucleotide insertions or deletions at the crossover sites. We constructed a genome-wide recombination map for each of the CTV strains in FS2-2 by aligning the recombinant sequences and contigs to the respective genomes. These maps revealed wide-spread recombinational activities at a genome scale that have not been reported previously. The recombinant sequencing reads were not uniformly distributed across the genomes. Most recombination events occurred at the 3' halves of the CTV genomes, with some recombination events taking place at the 5' halves of fs2_2_t30 and fs2_2_vt genomes. PARTICIPANTS: Zhongguo Xiong (PI): design and execution of the overall project and experiments; Caroline Coppinger, Megan Massey, Ben Walters, and Andrew Wang (undergraduate students): participation in various experiments during the project; William Dawson (collaborator): provided various CTV isolates from Florida; David Galbraith (collaborator): experimental design and analysis. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Citrus tristeza virus (CTV), and the diseases it causes, are an enduring threat to the U.S. and world-wide citrus industries. The impact of CTV infection spans from the destruction of South American citrus industries built on the sour orange rootstock in earlier 1900s, to present day limits on citrus production in many countries. In the U.S., stem-pitting isolates threaten all industries, and substantial costs are involved in disease management. The diversity of CTV and its interactions among different strains and with the citrus hosts constitute a complex pathosystem. Many intriguing facets of this pathosystem present challenges to formulating effective and informative management decisions of the tristeza disease: some CTV genotypes prevent superinfection of other CTV genotypes (cross-protection), while others co-exist in a complex; whereas sequences of a single, pure CTV genotype remain stable for long periods of time, new pathogenic isolates of CTV continue to emerge. Based on our recent findings, we hypothesize that the high level of promiscuous RNA recombination in CTV compensates for the extreme genome stability and the low mutation rate, and functions as a major force driving the production of genetic variants important for adaptation and evolution. Results from this project period indicate that divergent strains of CTV can co-exist in a single host. The genome of each strain contains an extremely low level of genetic diversity, one to two orders of magnitude lower than those reported for other RNA viruses. The observed low nucleotide diversities suggested that CTV has a low intrinsic mutation rate and, consequently, high genome stability. This is not entirely surprising, given the recent report that murine hepatitis coronavirus possesses a proof-reading mechanism that significantly lowers error rates during viral RNA replication. Because of the large genome size of closteroviruses and their genome similarities with those of coronaviruses, an as yet undiscovered but functionally similar proof-reading mechanism may also operate in closteroviruses to limit mutation rates during viral replication. Our results also suggest that CTV generates genetic diversity though promiscuous recombination. The recombinant sequence reads and recombinant contigs offer a glimpse of the viral genetic variability produced through recombination. This is the first time that genome-wide recombination has been characterized. Considering the frequency of recombination observed and the genome-wide distribution of recombination sites in the three CTV strains, we speculate that hundreds of thousands of recombinant genomes could be generated by recombination alone in the FS2-2 isolates. This large pool of genetic variants would provide sufficient starting materials for CTV to evolve and adapt rapidly to changing environmental. The role of recombination therefore appears far more important than we previously thought in the evolution of viruses such as CTV that maintain a low rate of error during RNA replication.
Publications
- No publications reported this period
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