Progress 09/01/20 to 08/31/23
Outputs
Target Audience:This award was to develop a virus-induced gene silencing vector to deliver small RNAs into the phloem of citrus that would function in various ways to help combat HLB disease. The target audience for this research was other citrus researchers, citrus growers, citrus regulatory agencies and scientific agencies. Also, since we were able to solve how to make VIGS vectors stable, the target audience was also investigators using virus vectors and those in the field of plant pathogens. Since the virus we were using is novel, with characteristics never before seen in plant viruses, the target audience is also virologists in general and specifically plant virologists. Since we discovered a new avenue for how viruses move in plants, researchers working on RNA movement were also a target audience. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This award has supported the training of 2 postdoctoral associates in the Simon lab, 1 postdoctoral associate in the Xiao lab, 2 research scientists at Silvec Biologics. 1 postdoctoral associate in the Vidalakis lab, and 1 undergraduate technician at Silvec Biologics. The team met 2- times a week over Zoom and the team received mentoring by Dr. Simon and Dr. Xiao in viruses and bacteria, research reporting, grant writing and project development. The undergraduate leaned about molecular biology techniques and working with plants. All senior individuals attended national meetings (post Covid) and presented their work. One postdoctoral associate from the Simon lab will soon be joining Silvec Biologics as a senior scientist and a second just joined USDA-Beltsville as a research scientist; 1 postdoctoral associate from the Vidalakis lab is now an assistant professor at Texas A&M. The remaining trainees are continuing to work on this important topic. How have the results been disseminated to communities of interest?Nearly all of the trainees attended national meetings in either Virology or Plant Pathology and presented this work. Dr. Simon also gave invited presentations to the California citrus board. Besides the papers reported here, one more paper is in the preparation stage on development of the VIGS vector and how it was stabilized. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
(Objective 1) Generate a CYVaV VIGS vector that serves as a universal platform for multiple, stable siRNA inserts. We have made excellent progress developing CYVaV into a VIGS vector. We determined how to stabilize inserted hairpins. Prior to this work, all plant virus vectors engineered to contain any foreign inserted sequence were unstable, even when inserts were small hairpins. Over time (usually days to a few weeks), viral progeny emerge in the population with most if not all of the inserted sequence deleted, frequently along with surrounding viral genomic sequences. We found that the conformation of the entire CYVaV1 genome has evolved to be metastable and highly interactive through canonical and non-canonical tertiary interactions, and natural hairpins throughout the dynamic genome structure have evolved to be perfectly tuned thermodynamically to maintain the genome-wide conformation required for critical virus functions. When a foreign (and thus non-optimized) hairpin is inserted, even into a location that does not discernably disrupt local secondary structure or any important functions, the hairpin is lost likely due to issues with the polymerase not smoothly engaged in replicating through the hairpin (i.e., the polymerase likely loses processivity leading to recombination events and deletions. We discovered that natural hairpins, or hairpins that "mimic" natural hairpins, are stable when introduced into locations that otherwise would pop out the inserted hairpin. We found that we could add hairpins up to 200 nt that would remain stable. Additionally, we also developed inserts that generated fusions with the RdRp ORF and would express peptides that could be cleaved off from the RdRp. However, as with CTV vectors, peptide levels are low as they are rapidly degraded. Currently, we working on peptide stabilization and have increased levels by at least 40-fold. (Objective 2) identify siRNAs that can target and kill CLas/L. crescens and incorporate the top one into the CYVaV vector. This objective was based on a paper uploaded to a preprint site that bacteria respond to siRNAs delivered either in vitro or in vivo. We have completed substantial work now towards this objective. We have found that a number of plant pathogenic bacteria are negatively affected by either non-specific siRNAs (P. syringae, Xylella fastidiosa, Liberibacter crescens), and/or specific siRNAs that target critical genes (P. syringae, Xylella fastidiosa, Erwinia amylovora) when added in vitro (E. coli was not affected). We have also delivered specific siRNAs by VIGS in planta and found a significant reduction in bacteria and pathogenesis for P. syringae, Xylella fastidiosa, and Erwinia amylovora. We have identified siRNAs that target genes in CLas that are known to be excellent targets for drug design. These are: (1) Bacterial DNA Gyrase and (2) Bacterial MurA, which catalyzes the first step in biosynthesis of the bacterial cell wall. Inactivation of the latter enzyme results in bacterial cell lysis and death. Using a VIGS vector co-infiltrated with Ti plasmid carrying the exact CLas genes, we have identified two siRNAs that reduce mRNA of the two expressed genes to near undetectable levels. We have been unable to infect N. benthamiana with L. crescens to test efficacy in vivo, and are currently growing papaya to conduct in planta experiments. Since we determined that bacteria require about 100-fold more siRNAs than fungi to achieve effects on gene expression, this may not be a viable approach. (Objective 3) identify siRNAs that can reduce expression of the Nicotiana benthamiana callose synthase gene (CS 7-like) and the orthologue from citrus (CscalS7) that are responsible for producing callose in sieve elements in response to pathogens. The goal of this objective was to identify an siRNA that can specifically target conserved sequences in CS7 from N. benthamiana and citrus, with a reduction of about 40 to 50% (this is still an essential gene). A 300 bp fragment that targets near identical sequences in N. benthamiana and citrus was cloned into a Tobacco rattle virus VIGS vector ) TRV2 (what we use to identify functional siRNAs before delivery to CYVaV. Systemic infection of the VIGS vector achieved about 75% silencing of the endogenous N. benthamiana CS7 gene. Silencing using a reduced 64 bp fragment cloned into TRV2 silenced about 50% of CS7 mRNA from N. benthamiana. This objective was significantly altered when papers came out indicating that targeting callose synthase actually had the opposite effect on the size of sieve element pores. We also determined that CYVaV uses phloem protein PP2 as a movement protein, the first plant virus to do so. PP2 is the protein that is induced upon pathogen infection, causing it to polymerize and clog the sieve tubes. When CYVaV is added to sap containing polymerized PP2, we discovered that de-polymerization occurs and small nodules form, which correlates with protection of the virus from treatment with RNase. Thus, simple infection by the wild-type vector may be associated with loss of symptoms (since phloem blockage should be reduced) and recent results are consistent with this hypothesis (this work is continuing). We have determined that a 300 nt fragment of the virus binds to PP2 much more efficiently than the full-length viral RNA and are currently examining if simple addition of this fragment will unblock the phloem in HLB-infected plants. (Objective 4) continue to identify siRNAs that can target all known isolates of CTV and prevent CTV infection of N. benthamiana, and siRNAs that accomplish the same goal with CVEV. We have generated CYVaV vectors with siRNAs that are stable and prevent CTV infection of N. benthamiana. We currently have Mexican lime infected with the vector and have grafted in CTV. We are continuing to monitor these plants to see if CTV levels are reduced over time. We have made major discoveries about the ORFs of CYVaV relatives that have dramatically changed our hypothesis about the presence and requirement for helper viruses. CYVaV is the only "Class 2" umbra-like virus that has lost ORF5. We have determined that ORF5 encodes a coat protein and not a movement protein as previously thought (all plant viruses except for these encode movement proteins or require assistance of a helper virus). Since most of these ULVs are known to NOT be associated in infected plants in the presence of a potential helper virus (which are required for umbraviruses), our current hypothesis is that Class 2 ULVs are independent infectious viruses (encoding a polymerase and a coat protein) that use host protein PP2 as a movement protein. This explains why helper viruses are not associated with this class of ULV. CYVaV has deleted two regions of its genome and no longer generates the coat protein. Therefore CYVaV is trapped in any plant that it infects. We have conducted 2.5 years of field and greenhouse studies on aphid transmission and no transmission by aphids has been observed. In plants co-infected with CVEV and CYVaV, aphids are able to transmit CVEV but CYVaV is not co-transmitted. Therefore, there is no longer a need to target CVEV. (Objective 5) Work out conditions for re-entry of CYVaV into citrus. This was by far the most important objective. We are now able to infect citrus using agroinfiltration or by dodder transfer. In infected plants 1.5 years old, 25 leaves were examined and all contained detectable amounts of CYVaV by q-RT-PCR. We also have mother trees and have developed simple techniques for graft transmission into seedlings.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2024
Citation:
Ying, X., Liu, J., Gao, F., Yang, S., Bera, S., and Simon, A.E. Independent systemic infection of umbravirus-like viruses in the absence of an encoded movement protein
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Reyes-Proa�o, E.G., Mendoza, A., Margaria, P., Menzel, W., Bera, S., Simon, A.E., Quito-Avila, D.F. 2022. Two new umbravirus-like associated RNAs (ulaRNAs) discovered in maize and Johnsongrass from Ecuador. Arch Virol 167, 2093-2098 doi: 10.1007/s00705-022-05525-4
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Liu, J., and Simon, A.E. 2022. Identification of Novel 5? and 3? Translation Enhancers in Umbravirus-like Coat-Protein-Dependent RNA Replicons. J Virol 96, e0173621
- Type:
Book Chapters
Status:
Published
Year Published:
2023
Citation:
Simon, Anne E., M�kinen, K., Li, Y., and Verchot, J. (2022) Plant Viruses. In: Fields Virology, 7th edition. Ed. Peter Hawley. Lippincott Williams & Wilkins, Publishers.
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Progress 09/01/21 to 08/31/22
Outputs
Target Audience:This award was to develop a virus-induced gene silencing vector to deliver small RNAs into the phloem of citrus that would function in various ways to help combat HLB disease. The target audience for this research was other citrus researchers, citrus growers, citrus regulatory agencies and scientific agencies. Also, since we were able to solve how to make VIGS vectors stable, the target audience was also investigators using virus vectors and those in the field of plant pathogens. Since the virus we were using is novel, with characteristics never before seen in plant viruses, the target audience is also virologists in general and specifically plant virologists. Since we discovered a new avenue for how viruses move in plants, researchers working on RNA movement were also a target audience. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This award has supported the training of 2 postdoctoral associates in the Simon lab, 1 postdoctoral associate in the Xiao lab, 2 research scientists at Silvec Biologics. 1 postdoctoral associate in the Vidalakis lab, and 1 undergraduate technician at Silvec Biologics. The team met 2- times a week over Zoom and the team received mentoring by Dr. Simon and Dr. Xiao in viruses and bacteria, research reporting, grant writing and project development. The undergraduate leaned about molecular biology techniques and working with plants. All senior individuals attended national meetings (post Covid) and presented their work. One postdoctoral associate from the Simon lab will soon be joining Silvec Biologics as a senior scientist and a second just joined USDA-Beltsville as a research scientist; 1 postdoctoral associate from the Vidalakis lab is now an assistant professor at Texas A&M. The remaining trainees are continuing to work on this important topic. How have the results been disseminated to communities of interest?The PI has given talks at major universities and at the American Society of Virology What do you plan to do during the next reporting period to accomplish the goals?We will continue to examine the need for a helper virus and continue to work on stabilization of the VIGS vector and on peptide expression.
Impacts
What was accomplished under these goals?
(Objective 1) Generate a CYVaV VIGS vector that serves as a universal platform for multiple, stable siRNA inserts. We have made excellent progress developing CYVaV into a VIGS vector. We determined how to stabilize inserted hairpins. A critical, unresolved issue was that plant virus vectors engineered to contain any foreign inserted sequence were unstable, even when inserts were small hairpins. Over time (usually days to a few weeks), viral progeny emerge in the population with most if not all of the inserted sequence deleted, frequently along with surrounding viral genomic sequences. We found that the conformation of the entire CYVaV1 genome has evolved to be metastable and highly interactive through canonical and non-canonical tertiary interactions, and natural hairpins throughout the dynamic genome structure have evolved to be perfectly tuned thermodynamically to maintain the genome-wide conformation required for critical virus functions. In other words, natural hairpins emanating from the genome "backbone" evolved to maintain a critical genome-wide metastable conformation. When a foreign (and thus non-optimized) hairpin is inserted, even into a location that does not discernably disrupt local secondary structure or any important functions, the hairpin is lost likely due to issues with the polymerase not smoothly engaged in replicating through the hairpin (i.e., the polymerase likely loses processivity leading to recombination events and deletions. We discovered that natural hairpins, or hairpins that "mimic" natural hairpins, are stable when introduced into locations that otherwise would pop out the inserted hairpin. We found that we could add hairpins up to 200 nt that would remain stable, and we could add them to several locations and stability was maintained. (Objective 2) identify siRNAs that can target and kill CLas/L. crescens and incorporate the top one into the CYVaV vector. This objective was based on a paper uploaded to a preprint site that bacteria respond to siRNAs delivered either in vitro or in vivo. We have completed substantial work now towards this objective. We have found that a number of plant pathogenic bacteria are negatively affected by either non-specific siRNAs (P. syringae, Xylella fastidiosa, Liberibacter crescens), and/or specific siRNAs that target critical genes (P. syringae, Xylella fastidiosa, Erwinia amylovora) when added in vitro (E. coli was not affected). We have also delivered specific siRNAs by VIGS in planta and found a significant reduction in bacteria and pathogenesis for P. syringae, Xylella fastidiosa, and Erwinia amylovora. We have identified siRNAs that target genes in CLas that are known to be excellent targets for drug design. These are: (1) Bacterial DNA Gyrase and (2) Bacterial MurA, which catalyzes the first step in biosynthesis of the bacterial cell wall. Inactivation of the latter enzyme results in bacterial cell lysis and death. Using a VIGS vector co-infiltrated with Ti plasmid carrying the exact CLas genes, we have identified two siRNAs that reduce mRNA of the two expressed genes to near undetectable levels. We have been unable to infect N. benthamiana with L. crescens to test efficacy in vivo, and are currently growing papaya to conduct in planta experiments. Since we determined that bacteria require about 100-fold more siRNAs than fungi to achieve effects on gene expression, this may not be a viable approach. (Objective 3) identify siRNAs that can reduce expression of the Nicotiana benthamiana callose synthase gene (CS 7-like) and the orthologue from citrus (CscalS7) that are responsible for producing callose in sieve elements in response to pathogens. This objective was significantly altered when papers came out indicating that targeting callose synthase actually had the opposite effect on the size of sieve element pores. We also determined that CYVaV uses phloem protein PP2 as a movement protein, the first plant virus to do so. PP2 is the protein that is induced upon pathogen infection, causing it to polymerize and clog the sieve tubes. When CYVaV is added to sap containing polymerized PP2, we discovered that de-polymerization occurs and small nodules form, which correlates with protection of the virus from treatment with RNase. Thus, simple infection by the wild-type vector may be associated with loss of symptoms (since phloem blockage should be reduced) and recent results are consistent with this hypothesis (this work is continuing). (Objective 4) continue to identify siRNAs that can target all known isolates of CTV and prevent CTV infection of N. benthamiana, and siRNAs that accomplish the same goal with CVEV. We are suspecting that CVEV is not a helper virus for CYVaV. We are unable to show specificity for packaging of CYVaV in the CP of CVEV. We have also been unable to show that the additional ORF (lost in CYVaV) that is present in all other relatives of CYVaV is a movement protein. We will be conducting experiments to show whether it is, in fact, a CP. (Objective 5) Work out conditions for re-entry of CYVaV into citrus. This was by far the most important objective. We are now able to infect citrus using agroinfiltration or by dodder transfer. In infected plants 1.5 years old, 25 leaves were examined and all contained detectable amounts of CYVaV by q-RT-PCR. We also have mother trees and have developed simple techniques for graft transmission into seedlings.
Publications
|
Progress 09/01/20 to 08/31/21
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This award has supported the training of 2 postdoctoral associates in the Simon lab, 1 postdoctoral associate in the Xiao lab, 2 research scientists at Silvec Biologics and 1 undergraduate technician at Silvec Biologics. The team met 3-4 times a week over Zoom and the team received mentoring by Dr. Simon and Dr. Xiao in viruses and bacteria, research reporting, grant writing and project development. The undergraduate leaned about molecular biology techniques and working with plants. 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?We will continue to work on each of the objectives, especially getting the vector to accept additional stable inserts. We will see if CYVaV can infect papaya, and use this system to examine effectiveness of siRNAs against L. crescens. We will also infect a number of citrus trees with CYVaV targeting CTV, to see if trees are immune to CTV infection. We will also determine if there is a detectable difference in callose staining in sieve elements when CYVaV has the insert targeting CS-7. Finally, we will continue to search for better siRNAs targeting CVEV, the presumptive helper virus for CYVaV. We will also be exqmining two additional possibilities for vectors that are derived from CYVaV: Defective RNAs, that are commonly generated and have a large deletion (and where translation of a functional RdRp does not occur) and dimers of CYVaV. Both of these molecules replicate and move systemicallly in N. benthamiana (the D-RNA in the presence of CYVaV). Both of these RNAs should be less reliant on a strict RNA conformation and should be much more ameanable to inserts, particularly large inserts.
Impacts
What was accomplished under these goals?
(Objective 1) Generate a CYVaV VIGS vector that serves as a universal platform for multiple, stable siRNA inserts. We have made excellent progress developing CYVaV into a VIGS vector. For this objective, we first needed to enhance the infectivity of our laboratory host, Nicotiana benthamiana. Previously, we could only achieve about 10% infectivity. We have now switched from syringe inoculation to a new protocol that uses vacuum infiltration (among other changes) and we routinely get 80-100% infectivity. We have also made progress stabilizing vectors that contain inserts. We determined that the issue with stability stems from a detrimental effect on translation, which is caused by structural changes throughout the CYVaV backbone vector that impact a novel bi-segmented ribosome recoding element among other translation elements. To help mitigate this affect, we have altered the vector to contain additional mutations that stabilize the active structure of the recoding element and destabilize the inactive conformation. This enhances translation of CYVaV with a previously unstable insert to near wild-type levels and consequently substantially enhances the stability of the insert. The mutations are also designed to destabilize the wild-type CYVaV, so that loss of an insert will produce a less fit vector, and it will be lost from the host over time. We have also found that replacing a particular 3'UTR hairpin with an siRNA insert that mimics its overall structure and stability leads to a very stable insertion. We are now combining our successes as we increase the number of inserts in the vector. (Objective 2) identify siRNAs that can target and kill CLas/L. crescens and incorporate the top one into the CYVaV vector. This objective was based on a paper uploaded to a preprint site that bacteria respond to siRNAs delivered either in vitro or in vivo. We have completed substantial work now towards this objective. We have found that a number of plant pathogenic bacteria are negatively affected by either non-specific siRNAs (P. syringae, Xylella fastidiosa, Liberibacter crescens), and/or specific siRNAs that target critical genes (P. syringae, Xylella fastidiosa, Erwinia amylovora) when added in vitro (E. coli was not affected). We have also delivered specific siRNAs by VIGS in planta and found a significant reduction in bacteria and pathogenesis for P. syringae, Xylella fastidiosa, and Erwinia amylovora. We have identified siRNAs that target genes in CLas that are known to be excellent targets for drug design. These are: (1) Bacterial DNA Gyrase and (2) Bacterial MurA, which catalyzes the first step in biosynthesis of the bacterial cell wall. Inactivation of the latter enzyme results in bacterial cell lysis and death. Using a VIGS vector co-infiltrated with Ti plasmid carrying the exact CLas genes, we have identified two siRNAs that reduce mRNA of the two expressed genes to near undetectable levels. We have been unable to infect N. benthamiana with L. crescens to test efficacy in vivo, and are currently growing papaya to conduct in planta experiments. A grant proposal is being prepared to look more deeply into targeting bacteria with siRNAs. (Objective 3) identify siRNAs that can reduce expression of the Nicotiana benthamiana callose synthase gene (CS 7-like) and the orthologue from citrus (CscalS7) that are responsible for producing callose in sieve elements in response to pathogens. The goal of this objective was to identify an siRNA that can specifically target conserved sequences in CS7 from N. benthamiana and citrus, with a reduction of about 40 to 50% (this is still an essential gene). A 300 bp fragment that targets near identical sequences in N. benthamiana and citrus was cloned into a Tobacco rattle virus VIGS vector ) TRV2 (what we use to identify functional siRNAs before delivery to CYVaV. Systemic infection of the VIGS vector achieved about 75% silencing of the endogenous N. benthamiana CS7 gene. Silencing using a reduced 64 bp fragment cloned into TRV2 silenced about 50% of CS7 mRNA from N. benthamiana. We are currently cloning the 64 nt sense fragment from CS7 into WT CYVaV iRNA vector for silencing in N. benthamiana and citrus plants. (Objective 4) continue to identify siRNAs that can target all known isolates of CTV and prevent CTV infection of N. benthamiana, and siRNAs that accomplish the same goal with CVEV. We have identified a highly specific siRNA that targets all known isolates of CTV and prevents CTV infection when delivered by CYVaV into co-infiltrated N. benthamiana. Since the insert at its current location does not support complete stability (defined as NO detectable reversion after 4-5 months in N. benthamiana), we are working on enhancing its stability as described in Objective 1. CVEV has been more difficult to target because of the difficulty of working with this poorly infectious virus (why it is such a rare virus in nature). We were unable to generate a version that expressed GFP at a high enough level, which would have simplified these experiments. Nevertheless, we have identified one siRNAs targeting CVEV that reduces virus levels by 71%. Since CVEV infects so poorly, we have not been able to determine yet if we can prevent infection. These experiments are continuing. (Objective 5) Work out conditions for re-entry of CYVaV into citrus. This was by far the most important objective. We have been working on many parallel approaches including infection of protoplasts and regeneration, transformation of citrus and regeneration, and packaging in coat protein of CVEV and delivery by stem peeling and dodder. I am please to report that packaging in the coat protein of CVEV and delivery by dodder appears to be successful. The packaging was accomplished using a TMV vector expressing the coat protein of CVEV and co-infiltrating N. benthamiana leaves with CYVaV. Virion formation and packaging was confirmed by EM and PCR, respectively. One end of the dodder was placed in microcentrifuge tubes containing encapsidated CYVaV (and some other forms) and the other end connected to Mexican Lime stems. One month later CYVaV plus and minus strands were detected in nearby tissue, indicating infected tissue. We are waiting to detect systemic infection, which can take several months.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
3. Kwon, S-J, Bodaghi, S, Gadhave, KR, Tzanetakis, IE, Simon, AE, and Vidalakis, G. 2021. Complete nucleotide sequence, genome organization and comparative genomic analyses of citrus yellow-vein associated virus, an umbravirus-like associated virus-like RNA. Front Microbiol. https://doi-org.proxy-um.researchport.umd.edu/10.3389/fmicb.2021.683130
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
4. Liu, J., Carino, E., Bera, S., Gao, F., May, J.P., and Simon, A.E. 2021. Structural analysis and whole genome mapping of a new class of plant virus subviral RNAs: umbravirus-Like associated RNAs. Viruses 13, 646; https://doi.org/10.3390/v13040646.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2021
Citation:
1. Wang, X., Olmedo-Velarde, A., Larrea-Sarmiento, A., Simon, A.E., Kong, A., Borth, W., Suzuki, J.Y., Wall, M.M., Hu, J., and Mellzer, M. 2021. Genome characterization of fig umbra-like virus. Virus Genes, in press.
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