Progress 09/15/23 to 09/14/24
Outputs
Target Audience:The target audience is scientists and growers. Dr. Simon attended the HLB annual meeting in Riverside, CA in 3/2014. Dr. Needham and Jang reported their results at the annual meeting of the American Society of Virology Changes/Problems:As described in the report of accomplishments, the CY1 vector displayed a novel phenotype of systemic acquired susceptibility making it a problematic vector for citrus. Fortunately, we (Silvec) was able to acquire the license for the CTV vector and we have significantly improved its properties by stabilizing the inserted defensin sequences. This will be further tested in our newly acquired ECDRE award What opportunities for training and professional development has the project provided?This project resulted in the training of 5 post-doctoral fellows and the professional development of 5 professional scientists. How have the results been disseminated to communities of interest?Results were reported at the Annual HLB Meeting in Riverside, CA (2024) and the Annual Meeting of the American Society of Virology (2024). What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Determine if the ability of WT CYVaV to de-polymerize PP2 improves outcomes in CLas-infected trees- A greenhouse trial was initiated in July 2023 to investigate whether CY1 pre-inoculated into Mexico Lime plants could mitigate symptom progression of HLB. In this trial, ten CY1 pre-inoculated or healthy plants were challenged with HLB through feeding of CLas-positive psyllids. Three months after the pruning (6 months after the initial CLas exposure), a severe ion-deficiency-like phenotype was observed in CY1 pre-inoculated Mexico Lime plants compared to the control plants (Fig. 2A). Additionally, elevated CLas titers (Fig. 2B) and starch accumulation (Fig. 2C) were found for CY1 pre-inoculated plants. The unexpected enhanced levels of CLas in CY1-infected plants quite obviously meant that CY1 was a problematic VIGS vector. The ability of CY1 to enhance bacterial levels was unexpected, and similar results were found using Pseudomonas syringae. Stabilizing and delivery of anti-bacterial defensins for treatment of citrus greening- We hypothesized that SoDs are not stable in plants when expressed by phloem-limited viruses (and likely by other means) and are therefore not accumulating to levels sufficient to substantially reduce CLas titers, especially if virus levels fluctuate. Since peptides are signaling molecules and therefore naturally have very short half-lives in vivo (seconds to a few minutes), these finding are not surprising. Currently, medical researchers are augmenting AMP stability by incorporating scaffolds to increase AMP size, which is associated with greater stability. Since artificial additions to SoD2* would dramatically increase costs and time for regulatory approval, we decided on four other strategies to increase SoD2* peptide size. All constructs were made and tested in N. benthamiana first for insert stability. Agrobacteria tumefaciens was used to infiltrate T-DNA cassettes containing SoD2* (the single version in the first generation [Gen1] CTV product), 2xSoD2*, CmPP16-SoD2*, and p13-Sod2* into N. benthamiana leaves. At four days post-infiltration (dpi), total protein was isolated from the leaves and subjected to western blot analysis. As shown in Fig. 3A, the single SoD2* was only detected when blots were substantially overexposed. In contrast, the three SoD2* fusion derivatives were present at significantly higher abundance, about 100-fold higher for 2xSoD2* and 500-fold higher for Cmpp16-SoD2* and p13-SoD2*. All tested constructs demonstrated significantly more bacterial reductions that the single SoD*. Developing siRNAs to target bacteria in plants- Cross-kingdom siRNA-mediated gene silencing has been utilized to engineer plant resistance against fungi, insects and parasitic plants. However, it is unclear whether this technology can be applied for control of plant bacterial pathogens because of the lack of the RNA interference machinery in bacteria. Our research shows that small double-strand RNAs (sRNAs, 21-25 nt) can indeed induce bacterial gene silencing and further inhibit bacterial growth both in vitro and in vivo. Stabilizing hairpins in virus vectors for long-term delivery of siRNA and peptides in citrus- The retention of hairpins with appropriate thermodynamic properties for at least 13-wpi in N. benthamiana demonstrated the potential for long-term retention of inserted hairpins for VIGS field applications. To evaluate if designed hairpins can be maintained for the longer times that are required for infection of citrus, an appropriately designed hairpin was inserted into CY1 and the resultant construct was infiltrated into 1 yr old Citrus aurantifolia (Mexican lime) trees. At 30-months post-infiltration, RNA samples were collected from two locations in two trees (Fig. 4) and subjected to RT-PCR. The amplified products co-migrated with those of the parental construct and chromatograms from batch sequencing the RT-PCR products revealed no detectable variants. To determine if the inserted hairpin was also stable after passaging into new plants, scions from WT CY1- and CY1+hairpin-infected trees were grafted onto 5 new Mexican lime trees and tested at 12-months post-grafting. Four of the five trees maintained the insert for the combined infection time of 42 months. Apply LDH delivery system (double-layer clay sheets [BioClay] through foliar spray The goals of this objective were to test the possibilities of using BioClay and spray-on application to deliver plasmids for launching viruses in citrus. As a preliminary test of the LDH delivery system to citrus, we first analyzed the internalization of LDH nanoparticles with average size of around 30 nm labelled with fluorescein (LDH-FL) post foliar application on different species. Direct foliar spray with no adjuvant was determined not efficient, where LDH has only been taken up by stomata guard cells for N. benthamiana (Fig. 5A) or no visual uptake for citrus (Fig. 5B). The addition of a specific penetrant into formulation allows nanoparticles to enter the stomata more effectively. While we observed the uptake into N. benthamiana mesophyll cells (Fig. 5A), we saw no uptake for citrus beyond stomata guard cells and limited surrounding cells (Fig. 5B). Based on the above results we hypothesized that the waxy cuticle of citrus is the main barrier for foliar uptake of sprayed nanoparticles. The small size exclusion limit and hydrophobicity of the cuticle would likely hinder the nanoparticle suspension internalization into the leaves and subsequent uptake by the cells. To overcome the potential cuticle barrier to LDH-FL uptake into citrus leaf, we decided to explore the use of physical aids by inducing wounds using either with scalpel blades or pinned roller to compromise leaf surface cuticle. Both abrasion techniques resulted in a significantly improved uptake of LDH-FL, with fluorescence readily observed in both applied and surrounding cells (Fig. 4B). However, the more subtle technique of rubbing the leaf surface with carborundum powder, which was usually adopted for helping virus inoculate on many plant species, only introduced a small amount of uptake in citrus leaf (Fig. 5B). Another applicable approach to enhance nanoparticle uptake through foliar spray is through high-pressure spray (Fig. 5C). Pressurization of the nanoparticle suspension directly drove the LDH-FL being taken up by species with soft leaf surface such as N. benthamiana and tomato. However, the pressurized nanoparticles still need to flood through the stomata pathway for citrus and not effective for wheat leaves. In addition, we have tested the delivery efficacy of high-pressure spray of LDH on N. benthamiana to deliver a 6.1 kb GFP encoded plasmid. We have observed clear GFP expressions using 4 bars while no substantial signal can be identified with 2 bar pressure for spraying. Delivery through cut petiole allows direct access of LDH nanoparticles to vascular system and bypasses the cuticle barrier during surface application (Fig. 6A). Based on these results we next tested delivery of GFP encoding plasmid through petiole application (Fig. 6C). Our results suggest that the petiole applied LDH nanoparticles is potentially the best mechanism for intact plant delivery or vascular tissue delivery vector. We tested the efficacy of delivery of two model viruses cloned into plasmids, 35S:PVX-GFP and 35S:CMV to N. benthamiana and tomato through petiole application (Fig. 7A and B). The successful delivery of PVX-GFP and CMV plasmid and expression of virus are also demonstrated on tomato, which is confirmed by RT-qPCR (Fig. 7E and 7). The systematic existence of both PVX-GFP and CMV mRNA was recorded from applied petiole to upper branches and the seedling shoot tip, indicating efficient delivery and self-propagation of expressed virus throughout the plants.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Ying, X., Bera, S., Liu, J., Toscano-Morales, R., Jang, C., Yang, S., Ho, J., and Simon, A.E. 2024. Umbravirus-like RNA viruses are capable of independent systemic plant infection in the absence of encoded movement proteins. PLoS Biol 22:e3002600
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Simon, A.E., Quito-Avila, D.F., and Bera, S. 2024. Expanding the plant virome: umbra-like viruses use host proteins for movement. Annu Rev Virol doi: 10.1146/annurev-virology-111821-122718
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
4. Johnson, P.Z., Needham, J.M., Lim, N.K., and Simon, A.E. 2024. Direct nanopore RNA sequencing of umbra-like virus-infected plants reveals long non-coding RNAs, specific cleavage sites, D-RNAs, foldback RNAs, and temporal- and tissue-specific profiles. Nucleic Acids Res Genomics Bioinform 6
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Progress 09/15/22 to 09/14/23
Outputs
Target Audience:The goal of this project is to develop tools with which to control HLB disease in citrus. The target audience for this research is other citrus researchers, citrus growers, citrus regulatory and scientific agencies. Also, since we are using virus-induced gene silencing (VIGS) vectors, the target audience is also investigators in the field of plant pathogens and plant viruses. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project involves four postdoctoral scientists among the professional scientists. These four postdocs have been extending their knowledge and learning about research connected to an important agricultural goal. They are also learning about conducting research that is highly collaborative. One undergraduate has also recently joined the project and is learning about conducting research. How have the results been disseminated to communities of interest?Two of the postdoctoral researchers presented their results at recent professional meetings. The PI, Anne Simon, has incorporated this research into her new undergraduate course on RNA. She also gave talks at the CRB annual meeting as an invited speaker and spent many hours talking to growers about the technology. From Silvec Biologics, the CEO, Dr. Rafael Simon, recently met with major citrus growers in Florida to update them on our progress. What do you plan to do during the next reporting period to accomplish the goals?We will be continuing with each of the projects outlined in the progress report. 1. We should learn about whether WT CY1 can by itself help to mitigate symptoms in citrus (an organic approach) 2. We should finalize the identification and testing of the anti-bacterial siRNAs (in N. benthamiana) 3. We should be able to test if knockdown of Mlo6 susceptibility gene helps to control CLas levels in infected citrus 4. We should be able to test if addition of the microRNA enhances the citrus immune system (looking at isolated leaves) 5. We should be able to test if peptides generated from CY1 vectors are anti-bacterial in N. benthamiana (and possibly citrus)
Impacts
What was accomplished under these goals?
[Please note that the start of the award was delayed until 3/1/23 due to issues at the University of Maryland with a facility use agreement] Identification of siRNAs targeting CLas: We have previously reported that siRNAs targeting essential genes showed non-specific inhibitory effects at high concentrations (> 3µM) for bothPseudomonas syringae(Pst) andLiberibacter crescens(Lcr)in vitro. Recently, we observed gene-specific inhibitory effects at lower siRNA levels (1.5 µM) targeting ADK and Gyrase A for both Pst and Lcr, which is consistent with in vivo assays using VIGS vectors inN. Benthamiana. In addition to ADK and Gyrase A, more target genes were selected from an essential gene list for bacteria survivalwere selected to test siRNA efficiency for bacteria growth inhibition using both Pst and Lcr, including ACP, GatC, Fba, DnaA, YchF, and BamA. In vitro assay results show that siRNAs targeting GatC can efficiently inhibit Pst growth but not Lcr while targeting Fba could inhibit Lcr growth but not Pst. Those results indicate that different bacteria could have different sensitivity to siRNAs targeting the same genes. Three regions in the Gyrase A gene of Clas have been screened for RNAi efficacy against transient expression of the Clas-GyrA gene using the TRV-based VIGS in vivo RNAi assay in infiltrated leaves. While all three fragments were able to decrease Clas-GyrA mRNA levels, one fragment (fragment 2) was far more potent for silencing and RT-PCR of GyrA mRNA was nearly undetectable. Therefore, two CY1 constructs carrying the siRNA inserts targeting fragment 2 were designed and constructed. We are currently acquiring the permits to be able to test for effects against CLas in excised leaves of infected citrus. We are also testing CY1 vectors carrying the three fragments for RNAi efficacy against transient expression of Clas-GyrA gene in N. benthamiana plants. Test if siRNAs targeting citrus mRNAs can strengthen the tree immune system In addition to targeting bacterial genes using CY1 VIGS, we are also taking the additional approach of generating higher levels of a natural micro RNA (miR395) that has been reported to enhance a plant's immune system by targeting the sulfate regulating genes APS1, which regulates sulfate accumulation, and SULTR2-1, which regulates sulfate distribution (loss of these increase sulfate levels). For production of miR395, four CY1 VIGS constructs were generated using citrus and A. thaliana pre-miRNA scaffolds. Following infection of N. benthamiana, we found that one construct (V1.1) generated 690 times more miR395 than mock or CY1 WT controls. The other three are currently being assayed. We have inoculated CY1-V1.1 into 18 N. benthamiana plants by agroinfiltration and assayed positive plants by qPCR for APS1 levels, finding that there was an 8% reduction compared to CY1 WT control levels. We are currently assaying the remaining three constructs for APS1 levels. We will subsequently assay all four constructs by qPCR for SULTR2-1 levels. We are also in the process of inoculating all 4 constructs into18 N. benthamiana plants and symptomatic leaves will be challenged with 1:1000 dilution of Xanthomonas campestris var vesicatoria (Xcv). Xcv (and CLas) lacks a CysZ transporter and as such should be sensitive to the elevated sulfate levels. Test if siRNAs targeting susceptibility gene Mlo6 can reduce bacterial (and fungal) infections A recent approach we are taking is based on reports that targeting susceptibility genes offers an alternative that is more durable than addition of resistance factors. We have developed siRNAs that target the Mlo6 gene, which confers resistance to powdery mildew and also other fungi and bacteria. Mlo6 is induced by pathogens as the encoded enzyme reduces salicylic acid levels by modifying its structure. One of three siRNAs developed to target Mlo6 reduced gene expression by 50% (complete downregulation via CRISPR can be harmful to plants), which correlated with a 50% decrease in fungal disease symptoms and a 10-fold decrease in Pst levels. We have constructed CY1 VIGS vectors containing siRNAs targeting the same region in citrus Mlo6 and have infected citrus with the construct. We are waiting for the plants to grow up and amplify them by grafting before they can be directed tested by the Hekt group for effect against CLas. Nanocarriers for introduction of CY1 into orchards We have determined an initial formulation of the BioClay nanosheets that, along with an adjuvant, penetrates into citrus through the leaf (both with an incision and in an intact leaf). We have also succeeded in introducing 35S-GFP plasmids into roots and getting expression in cells. We are working on optimization and will be testing introduction using high pressure sprays into leaves in the near future. Peptide expression using CY1 Because of the efficacious peptides against CLas that have been developed by others, we have been working on peptide expression by CY1, which normally only has two ORFs and no subgenomic RNAs. We have been successful at making stable constructs containing ORF2 (the RdRp) fused to a T2A cleavage sequence followed by a peptide ORF. The T2A sequence is nearly 70% effective in vitro and we can detect RdRp lacking the fused peptide in plants. To maximize expression, we have been successful in introducing an sgRNA promoter upstream of the fused peptide so that the peptide can also be generated from the sgRNA. We are currently making a construct with a peptide sequence acquired from the Hekt lab that has high anti-CLas activity. Can WT CY1 clear veins of CLas-infected citrus and improve plant outcomes? This objective is based on our finding that the mere presence of CY1 can depolymerize phloem protein 2 (PP2), which is thought to play a major role in the obstruction of sieve tubes upon CLas infection (the cause of root die-back). PP2 levels increase upon pathogen infections and high levels polymerize into stringy material, which is absent in N. benthamiana plants infected with CY1. Also, addition of CY1 to cucumber sap causes a visible loss (by EM) of the stringy proteinaceous material. To test the effects of WT CY1 on infected citrus, 13 healthy and 10 CY1-infected Mexican lime trees were delivered to Cornell (lab of collaborator Michelle Hekt) on 06/29/2023. All trees were challenged a month later using CLas-positive psyllids and we are waiting for symptoms to appear on (hopefully only) CY1-absent plants. In addition to CLas symptom assessment, we will also compare CLas infection rate as well as the average CLas bacteria titers in the CLas-infected plants. (Note that we do not expect any reductions in bacteria titers.) We have also made progress in understanding more about the biology of CY1, which is necessary for the approval process. A close relative of CY1 (CY2 from hemp) has the additional ORF (ORF5) found in all other members of Class 2 ulaRNAs. We have determined that ORF5 likely encodes a movement protein in the 30 kDa class of movement proteins (some capsid proteins also resemble these movement proteins). We have also determined that while the presence of ORF5 speeds up the initial infection of the ulaRNA, CY1 is more fit over time and by 6-weeks post-infiltration of both together, little CY2 can be found in the co-infiltrated plants. We have also explored the possibility that PP2 is the movement protein for CY1. CY1 VIGS vectors targeting the major PP2 species in the phloem is no longer able to efficiently infect N. benthamiana plants. CY2 altered to no longer be able to produce ORF5 is still able to infect N. benthamiana, but infection is delayed. This suggests that both CY1 and CY2 can make use of PP2, but the presence of ORF5 product supports more rapid initial systemic infection.
Publications
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