Progress 10/01/19 to 09/30/20
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
From March to September 2020, the accomplishments were smaller than originally anticipated notably due to the Covid19 pandemic. Nevertheless, they were important accomplishments, and meaningful results were made to allow the project to move forward as planned. Successful insect vector transmission of plant viruses requires a confluence of specific contributions from the virus, the insect and the plant. The many layers of complexity in the interactions among the participating members pose a major challenge that makes the absolute control of insect vector transmitted plant viruses difficult to achieve. In this context, it is of important relevance to pursue innovative, translational, and even unconventional ways of tackling the seemingly intractable problems by gaining insights into the basic biology of viruses and their interactions with plants and insect vectors. Notwithstanding their devastating impacts, plant viruses may be "subdued" and transformed into useful tools that benefit agriculture. Indeed, biotechnology has turned the tide on some viruses, allowing them to be engineered into benign, self-replicating genetic elements that can be used for various functions such as the production of protein products that plants can use directly, or products that can be purified for applications that benefit humanity, such as edible vaccines. It is in this framework that this project has been undertaken, the goal of which is to explore questions concerned with the fundamental (basic biology) and applied (biotechnology) aspects of several viruses in the family Closteroviridae. These questions are clearly reflected in the three main objectives of this project and the progress made during the current reporting period. Objective 1A - Studies were conducted to investigate the mechanism underlying the role of the LIYV minor coat protein (CPm) as a functional inhibitor of LIYV transmission. Achieving this objective requires being able to observe the LIYV CPm inhibitor in the foregut of the whitefly vector. As such, efforts were focused on two areas: 1) optimizing the labeling of E. coli expressed recombinant (r)CPm (the inhibitor) with biotin, and 2) performing foregut-retention assays in which fluorescence microscopy was used to examine whitefly vectors that fed on the biotinylated-rCPm and streptavidin conjugated quantum dot. Several repeated experiments that focused on these two areas were conducted, and the biotin-rCPm molar ratios and conditions for rCPm biotinylation were determined. The results produced promising indications that it would be possible to further optimize the labeling reactions to attain the desired levels of rCPm biotinylation. Objective 1B - Following last year's activity, in which we constructed three LCV-LIYV chimeric CPm mutants, we generated a fourth construct, LI1LC2A. This and the previous three constructs, LC1L12A, LC1LI2B, and LC1LI2C have since been renamed CPmP-1, CPmP-2, CPmP-3 and CPmP-4, respectively. The chimeric CPm of CPmP-1, -2, and -3 contain about 60%, 42%, 49%, respectively, of LCV CPm beginning after the methionine (encoded by the AUG start codon) at the N terminus. The LCV CPm sequences in these mutants are followed by LIYV CPm sequences, which make up the remaining 40%, 58% and 51% of the chimeric CPm of CPmP-1, -2, and -3, respectively. The chimeric CPm of CPmP-4 contains about 62% of LIYV CPm beginning after the methionine residue at the N terminus, and this is followed by 38% of the LCV CPm sequence. While all four mutants infected Nicotiana benthamiana plants systemically and expressed the expected chimeric CPms, CPmP-2, -3 and -4 were defective in retention and transmission by whitefly vectors (the Bemisia tabaci New World species); only CPmP-1 could be retained in the vectors' foreguts and transmitted at level comparable to that of wild type LIYV. Data from immunogold-labeling TEM and virion retention assays of CPmP-1 virions using anti-LCV CPm antibodies showed that the chimeric CPm was incorporated in an orientation that allowed the LCV CPm region to be recognized by the antibodies. In contrast, the same assays performed using anti-LIYV CPm antibodies could not identify the LIYV CPm region of the chimeric CPm. The upshot of these results is that the LIYV CPm exhibits novel organizational and functional plasticity in mediating virion retention in whitefly vectors. These results have opened new opportunities for further studies aimed at dissecting the mechanism of crinivirus transmission. Objective 1C - Studies were conducted to test the hypothesis that, like LIYV, LCV is also retained in the foreguts of the whitefly vectors that transmit it. The results were consistent with this hypothesis when foregut-retention and plant-to-plant transmission assays of LCV were conducted using the Middle East Asia Minor 1 (MEAM1) species of Bemisia tabaci whitefly vector. LCV retention and transmission was also performed using MEAM1 vectors that had acquired the virus in vitro by feeding on artificial sugar-based diet augmented with purified LCV virions. LIYV acquisition experiments performed using NW vectors and virions purified from a standard procedure involving the use of 2% triton at the extraction stage of the purification, had previously demonstrated that the acquired virions were retained in the vectors' foreguts. The foregut-retention of LIYV virions also corresponded with virus transmission when the virion-fed vectors were allowed inoculation feeding on target plants. In the case of in vitro acquired LCV virions purified by 2% triton, retention was observed in the foreguts of MEAM1 vectors, but it did not correspond with transmission in the same manner as observed for LIYV and NW vectors. No LCV transmission was observed when virions purified using 2% triton were acquired by MEAM1 vectors. However, foregut-retention transmission by MEAM1 was observed with LCV virions purified using 4% triton. This contrasted with the results showing that LCV virions purified by the 2% triton treatment were retained and transmitted by the NW vector (as reported in 2019). Thus, these results revealed that nuances in the retention and transmission of LCV virions by its whitefly vectors - B. tabaci NW and MEAM1 - exist and they could be influenced by a slight difference in the virion purification procedure. Objective 1D - We have not progressed beyond what was reported previously. We plan to perform the co-localization experiments in the next reporting period. Objective 2 - We have not progressed beyond what was reported last year. Plans are underway to perform the follow-up studies as stated in last year's report i.e. experiments to determine the viral RNA accumulation kinetics of the different constructs. Objective 3 - Following up from last year's studies, we have successfully engineered a bioactive and functional virus-based expression vector system that is constructed entirely from a California isolate of the T36 strain of CTV. In contrast to previous vector prototypes such as T36CA-GFP2 (constructed last year), this new vector system, named T36CA-V1.3, moves systemically in Citrus macrophylla plants as well as facilitate the sustained production of a genetic cargo using the green fluorescent protein as a proof of concept. T36CA-V1.3 differs from T36CA-GFP2 by four amino acids located in three different open reading frames encoding the p20 protein, the major coat protein (CP), and the p65 protein. The CP and p20 are known viral suppressors of RNA silencing while p65 is a putative CTV movement protein. These findings were included in a manuscript that was accepted for publication in a peer-reviewed journal (Chen et al, 2020; see products) in September 2020.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2020
Citation:
Chen, A.Y.S., Peng, J.H.C., Polek, M., Tian T., Ludman, M., F�tyol, K., Ng, J.C.K. 2020. Comparative analysis identifies amino acids critical for citrus tristeza virus (T36CA) encoded proteins involved in suppression of RNA silencing and differential systemic infection in two plant species. Molecular Plant Pathology 22(1):64-76.
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Progress 10/01/18 to 09/30/19
Outputs
Target Audience:Information obtained from the studies performed during this reporting period was disseminated to different target audiences - US and international scientists/researchers from academia, industry and government; outreach professionals, students, and stakeholders - through various channels including peer-reviewed publications, technical reports, seminars, academic and industry meetings, and laboratory visits. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
During the current reporting period, studies in Objective 1 have yielded new insights and tangible results that enabled us to move forward to the next stage of the project. Objective 1A -One biological replicate of the transmission blocking and virion retention experiment was performed to confirm the observations from earlier experiments. The outcome was consistent with the earlier results showing that when whitefly vectors were fed artificial diet containing a mixture of LIYV virions and LIYV recombinant minor coat protein (rCPm) expressed from E. coli: 1) no virus transmission was observed when the whiteflies were subsequently fed on virus-free plants, and 2) the absence of virus transmission corresponded with a drastic reduction in the number of vectors that retained virions in their foreguts. Also consistent was the finding that virus transmission was not inhibited in whiteflies that fed on artificial diet containing LIYV virions and LIYV recombinant major coat protein (rCP), and the number of whiteflies that retained virions in their foreguts was comparable to that of whiteflies that fed on virions alone. In this biological replicate experiment, we also included several technical replicates in which rCPm-mediated transmission blocking and virion retention were performed using a 10-fold increase of virion concentration. Transmission was observed in one technical replicate. However, we noted that in this instance, the corresponding number of whiteflies that retained virions in their foreguts was also significantly higher than those that were subjected to the same treatment, i.e. rCPm-mediated blocking using a 10-fold increase of virion concentration, in the other technical replicates but for which no corresponding transmission was observed. Taken together, our results are still consistent with the notion that rCPm can inhibit LIYV transmission when it is acquired from artificial diet (in vitro) along with LIYV virions. Objective 1B - Results were obtained for the studies on the transmission and virion retention of two sets of mutants. The first set of mutants were LIYV CPm mutants engineered with specific stop codon mutations. So far, none of these mutants were transmissible, and they were also poorly retained in the whitefly vectors. The second set of mutants were LIYV and LCV CPm domain swap mutants in which full-length LCV CPm was embedded in the LIYV CPm background in three different configurations. As with the stop codon mutants, none of these mutants were transmissible and there was a corresponding defect in virion retention in the vector's foregut (manifested by a reduction in the number of whitefly foreguts found to contain virions). We also constructed three LIYV and LCV CPm domain swap mutants containing different proportions of each CPm. These mutants, tentatively named LC1LI2A (mutant 1), LC1LI2B (mutant 2), and LC1LI2C (mutants 3), will be tested in the upcoming months for transmission and retention by whitefly vectors. Objective 1C - Experiments were conducted to demonstrate the retention of LCV within two whitefly (B. tabaci) species complexes: New World (NW) sp. and Middle East-Asia Minor 1 (MEAM1) sp. As reported previously, LCV was consistently transmitted by the NW sp. and this corresponded with the retention of virions in the vectors' foreguts. None of the vectors fed diet without virions showed fluorescent signals in the foregut (indicating no virion retention), and no transmission was observed. We have begun testing the retention and transmission of LCV virions using the MEAM1 sp. We anticipate performing three biologically replicated experiments in the coming months. Objective 1D - We have not progressed beyond what was reported previously i.e. the successful labeling of antibodies with specific fluorophores: anti-LCV CPm IgG (labeled with DyLight 488 or DyLight 594) and anti-LIYV CPm IgG (labeled with DyLight 488 or Alexa Fluor 594). We plan to perform the co-localization experiments in the next reporting period. Objective 2 - We engineered three reporter constructs using wild type (WT) M5gfp, a GFP-expressing defective RNA derived from LIYV R2, as the template. In M5gfp construct 1, the entire 3' NCR of LIYV R2 was replaced by that of LIYV R1. M5gfp constructs 2 and 3 both contained the 3' NCR of LIYV R2; in addition, they were augmented with the YSS of LIYV R1 at nucleotide positions 2555 and 2548, respectively. The in vitro produced transcripts of WT M5gfp or that of each of the three M5gfp constructs were co-inoculated with that of LIYV R1 to tobacco protoplasts to determine viral RNA synthesis and GFP production at 48 hours post inoculation (hpi). Green fluorescence was observed in protoplasts inoculated with LIYV R1 and WT M5gfp (the positive control) as well as LIYV R1 and M5gfp mutant 2. In contrast, no fluorescent signal was observed in protoplasts inoculated with either LIYV R1 and M5gfp construct 1 or LIYV R1 and M5gfp construct 3. More studies are underway to determine the viral RNA accumulation kinetics in the protoplasts inoculated with these different constructs. Objective 3 - Having obtained the complete genome sequences of two California strains, T36CA and T30CA, of CTV in the previous reporting period, we began the construction of the full-length cDNA clones (constructs) of these strains. These constructs were designed to contain the green fluorescent protein (GFP) coding sequence at one of several pre-determined locations in the CTV genome. If the constructs were infectious, the production of GFP would serve as a traceable marker for biological activity. In addition, GFP production would also serve as a proof of concept that CTV could be engineered to carry and express a genetic cargo in plants. During this period, we made a construct, T36CA-GFP2, in which the GFP coding sequence was engineered downstream of the p23 open reading frame (ORF) in T36CA. When this construct was inoculated to N. benthamiana plants by agrobacterium-mediated transient expression (agroinoculation), GFP expression was observed in the inoculated leaves but not the systemic leaves. We also conducted T36CA-GFP1 (engineered with the GFP coding sequence engineered in between the CPm and CP ORFs) to N. benthamiana plants and GFP expression was also observed in the inoculated leaves but not the systemic leaves. We also constructed two T30CA derivatives. The first derivative was one in which the CPm ORF was engineered with an intron from a potato gene. This was done to eliminate E. coli toxicity caused by the cloned T30CA sequences. The second derivative was engineered with two splice acceptor sites aimed at circumventing the lack of biological activity of T30CA in N. benthamiana plants. Both constructs did not show GFP expression in the locally inoculated leaves. We will continue to engineer more constructs and tests their biological activities in the next reporting period.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Wang H., Lei, T., Xia W., Cameron S., Liu, Y., Zhang Z., Gowda, M.M.N., De Barro, P., Navas-Castillo J., Omongo, C. A., Delate, H., Lee K., Patel M., Krause-Sakate, R., Ng, J., Wu, S., Fiallo-Olive, E., Liu, S., Colvin, J., Wang, X. Insight into the microbial world of Bemisia tabaci cryptic species complex and its relationships with its host. Scientific Reports 9:6568.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
D�der, B., Burgbuchler, M., Macia, J., Alcon, C., Curie, C., Gargani, D., Zhou, J.S., Ng, J.C.K., Brault, V., Drucker, M. Split green fluorescent protein as a tool to study infection with a plant pathogen, Cauliflower mosaic virus. PLoS ONE 13(3): e0213087.
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Progress 04/11/18 to 09/30/18
Outputs
Target Audience:Information obtained from the studies performed during this reporting period was disseminated to different target audiences - scientists/researchers from academia, industry and government; outreach professionals, students, and stakeholders - through various channels including peer-reviewed publications, technical reports, seminars, academic and industry meetings, and laboratory visits. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
This project focuses on the fundamental (biology and molecular biology) and applied (biotechnology applications) studies of specific viruses in the family Closteroviridae. By interrogating their interactions with plants and/or insect vectors that transmit them, we seek to better understand the roles that the viruses play in plant pathogenesis and disease transmission. We are also exploiting the knowledge gained from these studies for the development of molecular biotechnology systems aimed at enabling innovative strategies towards the effective management of pests and pathogens that pose significant threats to subtropical agriculture. The host-to-host transmission of viruses by plant feeding insects involves many layers of complex interactions occurring at the nexus of all participating partners i.e. the virus, insect and plant. Although our knowledge of virus transmission based on classical observational studies is quite broad, the precise mechanisms underpinning virus-vector-plant interactions mediating disease and disease transmission remain poorly understood. During the current reporting period, studies in Objective 1 have yielded new insights and tangible results that enabled us to move forward to the next stage of the project. In Objective 1A - a novel approach to block the whitefly (Bemisia tabaci) transmission of the crinivirus lettuce infectious yellows virus (LIYV; a member of the Closteroviridae) using recombinant LIYV capsid proteins - when whitefly vectors were fed artificial diet containing a mixture of LIYV virions and LIYV recombinant minor coat protein (rCPm) expressed from bacteria (E. coli), no virus transmission was observed when the whiteflies were subsequently fed on virus-free plants. The absence of virus transmission corresponded with a drastic reduction in the number of vectors that retained virions in their foreguts. In contrast, virus transmission was observed when virus-free plants were given access to whiteflies that fed on artificial diet containing LIYV virions and LIYV recombinant major coat protein (rCP), and the number of whiteflies that retained virions in their foreguts was comparable to that of whiteflies that fed on virions alone. These results are consistent with the CPm being a major determinant of LIYV transmission and support our hypothesis that rCPm can inhibit LIYV transmission when it is acquired from artificial diet (in vitro) along with LIYV virions. For Objective 1B - identification of molecular determinants mediating the specific retention (in the whitefly vector) and transmission of LIYV and lettuce chlorosis virus (LCV) - accomplishments include: 1) the introduction of specific stop codon mutations in the CPm of LIYV that would result in the production of truncated CPm by the mutant viruses, and 2) domain swap mutants made using the CPm of LIYV and LCV. More specific mutants are anticipated in the pipeline. Our plan is to study the whitefly retention and transmission of these CPm mutants throughout the course of this project. For Objective 1C - determine the vector retention sites of virions of other criniviruses - experiments were conducted to demonstrate the retention of LCV within two whitefly (B. tabaci) species complexes - New World (NW) sp. and Middle East-Asia Minor 1 (MEAM1) sp. Results from repeated experiments indicated that LCV was consistently transmitted by the NW sp. and this corresponded with the retention of virions in the vectors' foreguts. None of the vectors fed diet without virions showed fluorescent signals in the foregut, and no transmission was observed. For Objective 1D - determine whether LIYV and LCV virions co-localize to similar sites in the foregut regions of their common whitefly vector - we successfully labeled the following antibodies with specific fluorophores (indicated in parentheses): anti-LCV CPm IgG (DyLight 488 or DyLight 594) and anti-LIYV CPm IgG (DyLight 488 or Alexa Fluor 594). The fluorophore-labeled IgGs detected their respective target viruses that retained in the vector's foregut and did not cross-react with the non-target virus. The 3' terminus in the genome of RNA viruses contain secondary structures that, on their own, or in association with those located at proximal and/or distal regions of the genome, are involved in many critical functions including the initiation and regulation of RNA synthesis, translation regulation, and virion encapsidation. Insights into these viral RNA structure-mediated functions hold the potential for targeted approaches towards the control of viruses. However, neither structures nor functions of the 3' terminal genomic regions have as yet been thoroughly investigated for criniviruses in the family Closteroviridae. In Objective 2 - in vivo analysis of a highly conserved RNA structure in the 3' non-coding region (NCR) of the crinivirus genome involved in pathogenesis resulting from viral translation and/or concomitant viral RNA synthesis - we focused on an experimentally determined Y-shaped structure (YSS; located in the 3' NCR of genomic RNAs 1 [R1] and 2 [R2] of LCV) that exhibits viral RNA replication-associated translation activity. Similar YSS elements have been predicted in the 3' NCRs of all criniviruses. However, LIYV appears to be exceptional in that a YSS is predicted only in R1 but not in R2. Furthermore, LIYV exhibits asynchronous gRNA accumulation kinetics characterized by a 24-hour lag in the accumulation of R2 versus that of R1. We hypothesize that asynchronous gRNA accumulation is a consequence of the absence of a YSS in the 3' NCR of LIYV R2. To test this hypothesis we constructed specific 3' NCR mutants centering around or targeting the YSS. During this period, we made two constructs using the cloned infectious cDNA of LIYV R2 and that of M5gfp, a GFP-expressing defective RNA derived from LIYV R2. In both constructs, the 3' NCRs were replaced with that of LIYV R1. These constructs, along with several others that are in the construction pipeline, will be tested in tobacco protoplasts for the role of the YSS in influencing viral RNA accumulation kinetics. Our efforts in developing applications for improving citrus health and productivity involves the development of a molecular biotechnology toolkit that is built upon a citrus tristeza viral (CTV) vector platform. This CTV-based platform will be capable of delivering RNA interference (RNAi) and other defense-associated or trait improvement genes to citrus plants. In Objective 3 - strengthen on-going efforts in generating a functional Citrus tristeza virus (CTV)-based vector that can be developed into a biotechnology platform for controlling insect pest and pathogens, and for improving citrus traits - two CTV strains endemic to California were chosen as candidates for making cloned infectious cDNAs upon which the viral vectors will be built. Our initial efforts were focused on determining the full-length genomic sequences as well as nucleotides (nts) located at the terminal ends of the viral genomes. Specifically, the terminal nts were identified by sequencing cDNA clones of the plus- and/ or minus-strand of the viral double-stranded (ds) RNAs generated using 5' and 3' rapid amplification of cDNA ends techniques. Subsequently, cloned cDNAs corresponding to the complete genome sequences of both viruses were generated using reverse transcription-polymerase chain reactions, sequenced, and subjected to phylogenetic analysis. Our study revealed the extent of nt heterogeneity at the terminal ends of the genomes of these CTV strains. Pairwise comparisons and phylogenetic analysis with multiple reference sequences revealed that the genomes of both CTV strains were highly conserved with those of the respective strains isolated in other parts of the world. These findings have been vital in facilitating our ongoing efforts to construct the full-length cDNA clones of these virus strains.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Chen, A.Y.S., Watanabe, S., Yokomi, R., Ng, J.C.K. 2018. Nucleotide heterogeneity at the terminal ends of the genomes of two California Citrus tristeza virus strains and their complete genome sequence analysis. Virology Journal 15:141.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Zhou, J.S., Drucker, M., and Ng, J.C.K. 2018. Direct and indirect influences of virus-insect vector-plant interactions on non-circulative, semi-persistent virus transmission. Current Opinion in Virology 33:129-136.
- Type:
Other
Status:
Published
Year Published:
2018
Citation:
Peng, J., Chen, A.Y.S., and Ng, J.C.K. 2018. Fighting HLB with a Citrus tristeza virus (CTV)-based vector development of biologically active cDNA clones of CTV strains originating from California. Citrograph 9(3): 66-70.
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