Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 212 - Pathogens and Nematodes Affecting Plants | 999 - Citrus, general/other | 1040 - Molecular biology | 100% |
Huanglongbing (HLB, also known as citrus greening), one of the most destructive citrus diseases, is threatening the global citrus industry. After HLB spread in the US, HLB disease management-related expense reached 1.2 billion dollars annually. The most attractive and sustainable disease management is to develop resistance cultivars and/or to stimulate and utilize natural defense mechanisms of host plants. We aim to discover regulatory components that are involved in natural defense responses of tolerant citrus cultivars.Our ultimate goal is to develop HLB - resistance cultivars and/or efficient HLB management method.
Identification and expression validation of tolerant/resistant-specific sRNAs and target genes We identified one panel of sRNAs that were specifically down-regulated in HLB-positive tolerant US-942 but not in susceptible genetype Cleopatra. Many of these sRNAs target defense genes or regulatory signaling genes, such as PSL4, NDR1, NAC transcription factors, which are involved in plant defense responses against pathogen infections. These genes are upregulated in US-942 to promote host immunity against HLB, and therefore are thought to be appealing candidates for overexpression analysis in citrus. In addition, we also identified a panel of sRNAs that were induced specifically in US-942 in response to CaLas infection but not in Cleopatra. These sRNAs are likely target negative regulators of plant defense responses. Here, we will first validate the expression of these identified sRNAs and targets in US942 and Cleopatra, as well as Australia hybrids, Sydney hybrid. We will use transient functional assays and stable transformation approaches to assess the function of these identified US-942 specific sRNAs and their targets. Our preliminary data confirmed the expression of several selected sRNAs and their targets in both US-942 and Cleopatra on some of the candidates, and four sets of genes and targets were listed below for in-depth functional analysis. At the same time, we will continue to validate and examine more sRNA candidates. A.PSL4/EFR EFR is a leucine-rich repeat receptor kinase (LRR-RLK) that recognizes bacterial PAMP (pathogen associated molecular patterns) EF-Tu, and trigger basal defense responses against pathogen infection - so called PAMP triggered immunity (PTI). EFR is only found in Brassicaceae1, and requires PSL4 for its stable accumulation to establish robust immunity2.PTI contributes to basal and nonhost resistance, which is durable and broad-spectrum3. Introducing EFR to Solanaceous plant tomato, or even monocot wheat can enhance plant resistance to fungal and bacterial pathogens4 5. Here, we identified a sRNA that targets PSL4 was down-regulated only in HLB-positive US942, which led to PSL4 induction after CaLas infection. We propose to overexpress PSL4 together with EFR receptor in susceptible citrus variety to test whether they could truly enhance the host resistance against HLB, as found in wheat and tomato. We have set up the collaboration with Dr. James Thomson, from USDA Albany, CA to use his RMCE system for introducing multiple genes into the plants at the same time. His supporting letter was attached.B.siRNA-vad1/EIN2/NDR1 We also identified one up-regulated sRNA that targets VAD1 (vascular associated death1) and two down-regulated sRNAs that target EIN2 and NDR1, respectively. vad1 Arabidopsis mutant displays enhanced expression of defense genes, accumulation of high levels of SA, and increased resistance to virulent and avirulent strains of Pseudomonas syringae pv tomato (Pst). These responses of vad1 were dependent on EIN26, which was a negative regulator of C2H4 signaling pathway. Ethylene pathway is generally antagonistic to SA pathway. Up-regulation of the negative regulator of ethylene pathway EIN2 is likely to promote SA signaling pathway and contributes to HLB resistance. In addition, NDR1 is required for non-race specific resistance to bacterial and fungal pathogens through mediates systemic acquired resistance (SAR) response. Citrus NDR1 could functionally complement Arabidopsis ndr1 mutant7. We propose to overexpress the sRNA that targets VAD1 (sRNA-vad1) together with EIN2 and NDR1 in susceptible citrus variety, we hypothesize that this sRNA and these genes can promote overall plant natural defense responses against CaLas infection.C.siRNA-OLIGOPEPTIDE TRANSPORTER 1-like (OPT1-like)We identified a HLB-induced sRNA from US-942 that targets OPT1-like protein gene, which is responsible for metal homeostasis. OPT1-like protein was shown to be hijacked by bacterial pathogens to transport virulence factors into host cells. Down-regulation of OPT1-like gene may reduce the transportation of virulence factors from the bacterial pathogen. Therefore, we propose to over-express this sRNA.D.NAC45/86-dependent exonuclease-domain proteinA gene encodes NAC45/86-dependent exonuclease-domain protein was targeted by a HLB-down-regulated sRNA. It was highly induced in HLB-positive US942 (more than 8-fold induction). Overexpression of NAC45/86-dependent exonuclease-domain protein gene in Arabidopsis promotes sieve element maturation8. Because Calas is a phloem-limited bacterial pathogen, the phloem development and maturation are essential to ensure the transportation of proteins and nutrients. Therefore, we also propose to assess the function of this gene in producing more healthy phleoms after HLB infection.Approach 2: Functional analysis of candidate sRNAs and genes with Nicotiana benthamiana transient expressed systemWe have set up a transient expression assay Nicotiana benthamiana and routinely use it to assess the functions of sRNAs and genes in plant immune responses in Nicotiana benthamiana. We will generate binary constructs that expressing identified sRNAs and these target genes, and transiently express them in Nicotiana benthamiana. Various bacterial and fungal pathogens in the lab will be tested on the infiltrated leaves to see the defense response effect comparing to the empty vector inoculated plants. In addition, the defense marker genes will be tested to monitor the defense responses. Once the BL3 lab is in place (which is projected to be ready in a year time), we will also perform the infection of Candidatus Liberibacter solanacearum growth assay to evaluate their natural resistance response of these tested genes and small RNAs 9,10 (The Candidatus Liberibacter solanacearum work will be in collaboration with Dr. Manjunath Keremane, but it may be out of the scope of this one year time frame).Approach 3: Application of candidate sRNAs and genes as HLB resistance tools for susceptible citrus?The function of these sRNAs and genes in citrus defense against HLB will be finally tested in citrus by generating transgenic citrus plants. We will cooperate with Dr. James Thomson (USDA, ARS, PWA, WRRC-CIU) to produce stacked transgenic constructs for introducing multiple genes into the citrus plants. My postdoctoral fellow will go to Jim's lab and make the DNA constructs and generate Agrobacterial strains for transformation. James estimated the duration for this tasks would be 2 months. We will use the University of California, Davis facility to generate the transgenic citrus plants.
Target Audience
Scientific communities, biotech and agricultrual industry, growers and farmers.
Changes / Problems
Nothing Reported
Training & Professional Development
The graduate student in Jin lab is learning molecular biology techniques of sample testing and introduced the project in Dr. Khan's class (NASC093) which focused on citrus research and the industry. The project served as a basis for undergraduate research in the Jin group to understand SAMP function and regulation on controling HLB.One student performed work on an honors thesis project addressing these two research areas and another performed research for course credit.
Dissemination Streams
Dissemination of the research findings were published in high impact journals, which have been broadly read by not only the scientific communities, agricultural industry partners, but also the citrus growers. The PI Dr. Jin has been interviewed by many domestic and international news outlets, such as LA times, NPR news, Australian Broadcasting Corporation's national rural affairs TV program Landline, to introduce this finding. The PI Hailing Jin has given more than ten talks on this antimicrobial peptide for controlling citrus HLB at international conferences, universities, and to citrus industry (CRB, CRDF, and companies) and citrus growers. The postdocChien-Yu Huang in Jin group has joined virtual conferences to present our work at Plant Biology 2021 Worldwide Summit, North San Diego California Rare Fruit Growers (CRFG) event and San Diego Botanic Garden Monthly Volunteer Meeting.
Next Reporting Steps
Nothing Reported
Target Audience
Scientific communities, farmers and growers, and industry partners
Changes / Problems
Nothing Reported
Training & Professional Development
The project has thus far provided training to graduate andundergraduate researchers. The graduate student in Jin's lab have training on peptide expression and gene expression level evaluation. The project manager in Jin's lab manage the project progress and organized the meetings including the physical annual meeting on Jan 30th 2020 at Riverside, CA. Pending reopening of the campus, two additional undergraduates will participate in the project in Mauck Lab; one additional graduate student will join the project in Jin Lab.
Dissemination Streams
We have a the grant annual meeting in Riverside, CA at Jan 30th 2020 with advisory broad member invited and provide us important advised. The meeting agenda are attached. Dr. Mauck have incorporated lessons on HLB and ACP into introductory biology and introductory entomology courses taught on campus. The project manager Chien Yu Huang have a presentation at department seminar to introduce the HLB disease and our work to students. We have joined conference meeting to present our work to researchers and growers including: oral presentation at UCR Citrus Day for Industry. Riverside, CA at Feb 2019 which is host by Co-PI Dr. Kahn; oral presentation by zoom meeting to California Citrus Nursery Board at Feb 2019; Poster presented at International Research Conference on Huanglongbing VI. Riverside, CA (Co-PI Vidalakis is the major host); Oral presentation at 2019 IS-MPMI XVIII Congress. Glasgow, Scotland. Dr. Jin and Dr. Chien-Yu Huang attended the meeting, and both gave a presentation. Dr. Huang exclusively talked about the new progress of this project, and her talk was very well received by the international plant pathology community.
Next Reporting Steps
We will continue experiments with exposures and treatments and collect the data for the result analysis. <br><br>
<br>What was accomplished under these goals? For treating the infected tree, we've mainly focused on trunk injection method in the past year. We have finalized the concentration of SAMP and application frequency for treatments. To date, we have completed the following application of treatments at CRF, UCD: (note that all the citrus scion is grafted onto Carrizo rootstock and are seedling plants).We have tested different concentration of SAMP solutions on CLas-positve Citrus marophylla,Madam Vinous sweet orange, Lisbon lemon,and monitor the CLas titer for 20 months. We found thatthe CLas titer decreased after the second treatment in all three varieties,and remained low during theexperiments. The plants will be continously monitored for another 6 months. For testing the protection effect of SAMP, we have started a test on 20 one-year-old health Madam Vinous trees at CRF. The plants were subjected to ACP infestation (no choice feeding test)after foliar spray with buffer or SAMP. We have monitored the expression of defense marker genes and collected tissue for CLas titer detection. The trees growing better with SAMP spraying after 12 months of infection. We have observed the SAMP treatment plants have fewer ACP feeding marks, eggs and nymphs after 2 weeks ACP exposure. The plants with SAMP treatment have fewer infection plants and lower CLas titer after infection for 12 months. Expression of foreign genes using virus-based vectors is a strategy to protect plants against disease-causing pathogens. Citrus tristeza virus (CTV)-based vector allows transient expression of foreign proteins in citrus trees. Thus, expression of an anti-Huanglongbing (HLB) protein via CTV-based vector has a great potential to protect uninfected trees in the field and potentially cure trees with already established HLB infection. During this project period, a nucleotide sequence encoding the SAMP1 was cloned into the insertion site of the CTV-vector located at the 3' end of CTV genome, under the promoter of the natural CTV coat protein (CP) subgenomic RNA. The resulting construct was named as CTV-SAMP1. <br><br><b>Publications</b><br>
Target Audience
scientific communities, industry and growers
Changes / Problems
Nothing Reported
Training & Professional Development
This project has supportedone graduate student in Jin's lab, who hadtraining on peptide expression and gene expression level evaluation.
Dissemination Streams
We have joined multiple conference meetings to present our work to researchers and growers.
Next Reporting Steps
Everything is going on as planned, we expect to accompllish the goals in time as proposed. <br><br>
<br>What was accomplished under these goals? This project aims toDevelop therapies using a novel class of citrus-derived dual-functional antimicrobial peptides to cure HLB-positive trees and to protect healthy trees from infection. We have applied SAMP by trunk injection on HLB-positive trees of three different citrus varieties, and sampling at different time points to monitor the CLas titer and growth of the trees. We have also treated healthy sweet orange trees with SAMP solution, and then expose them to hot psyllids to monitor the progress of HLB symptom and CLas titers. These experiments are still on going, and the results will be reported in the next funding period. <br><br><b>Publications</b><br>
Target Audience
Scientific societies, agricultural industry and growers
Changes / Problems
Nothing Reported
Training & Professional Development
The Covid-19 pandemic significantly limited opportunities for professional development in most regards (cancelled meetings, few opportunities for in-person talks, and undergraduates banned from working in on-campus labs). However, with some creative thinking, we were able to develop remote projects for two undergraduate researchers in 2020. Godfrey group is learning new molecular biology techniques to improve testing of samples. This was done by working with other scientists at UC-Davis. Jin group have a new graduate student join the project. One student in Mauk group performed experiments evaluating effects of priming agents on psyllid behavior.
Dissemination Streams
We disseminated the results by publications, meeting presentations and talk with growers and industry.
Next Reporting Steps
We plan to finish the res proposed experiments as in the proposal, and there shouldn't be any delays. <br><br>
<br>What was accomplished under these goals? By comparative genetics and genomics analysis between HLB-sensitive cultivars and HLB-resistant/tolerant citrus hybrids and relatives, we identified and cloned a novel class of antimicrobial peptides (termed APs) that can effectively inhibit/kill multiple Liberibacter species, including CLas that causes HLB. We demonstrated that SAMP not only effectively reduced CLas titer and disease symptoms in HLB-positive trees but also induced innate immunity to prevent and inhibit infections. Importantly, unlike antibiotics, SAMP is heat stable, making it better suited for field applications. Spray-applied SAMP was taken up by citrus leaves, stayed stable inside the plants for at least a week, and moved systemically through the vascular system where CLas is located. We further demonstrate that SAMP is most effective on !-proteobacteria and causes rapid cytosol leakage and cell lysis. <br><br><b>Publications</b><br>
Target Audience
The PI reached different levels of the target audience, including acts or processes that deliver science-based knowledge to people through formal or informal educational programs. Examples include: formal classroom instruction, laboratory instruction, or practicum experiences; development of curriculum or innovative teaching methodologies; internships; workshops; experiential learning opportunities. The PI is also actively involved in extension and outreach activities.
Changes / Problems
Nothing Reported
Training & Professional Development
This project provided excellent training opportunities to an undergraduate student and a postdoctoral fellow. The postdoctoral fellow presented her work in the Citrus Research meetings, and Departmental seminar series. The postdoc was also trained on how to supervise undergraduate students.
Dissemination Streams
Yes, two research articles were published in highly respected journals. The PI and the postdoc have been giving talks to citrus growers. The PI also actively took part in the outreach program by giving presentations and discussions during press release events on citrus HLB, etc. ?
Next Reporting Steps
The project was well designed, and we are currently on the right track. We would expect to accomplish the goals as planned during the next reporting period. <br><br>
<br>What was accomplished under these goals? We have made significant progress on the investigation of the roles of small RNAs inregulating plant host defense against bacterial and fungal pathogens in citrus, tomato, and Arabidopsis. We also published two papers on the regulations of small RNAs upon bacterial and fungal infection (Huan Wang et al., New Phytologist, 2017, Ming Wang et al., RNA Biol. 2017). The unpublished findings and experimental results are listed below: For controlling citrus HLB, we have identified a list of potential plant defense regulators against HLB from small RNA profiling analysis compared between HLB-resistant/tolerant US942 and HLB-sensitive Cleopatra. Tables 1 and 2 show two short lists of HLB Defense Positive Regulators (HDPRs) and HLB Immune Suppressors (HISUs) to be used as tools to fight CLas. The expression level of selected candidate regulators displays a similar trend in two different HLB-tolerant varieties from different geographic and genetic backgrounds. Here we show examples of one sRNA, 942si2010, and its target gene, VAD, which is expected to act as an immune suppressor. Two examples of HDPRs, BRAP and HS, have higher expression levels in HLB-tolerant varieties. (Fig.1). Figure 1. The expression level of candidate regulators, small RNA 942si2010, VAD, BRAP, and HS, have consistent expression patterns in two different HLB-tolerant verities, US942 vs. Cleopatra and hybrids from Australian Eremocitrus and citrus sp. 72 vs. 74. The expression level is detected by qRT-PCR with Actin as the reference gene. Because we can't directly work on CLas in Southern California before our BSL3 facility is ready, we established Nicotiana benthamiana/Candidatus Liberabacter solanacearum (CLso) phytosystem. CLso is a closely related bacterial species of CLas, which can infect solanaceaous plants. To evaluate the function of candidate regulators, we performed Virus Induced Gene Silencing (VIGS) to knock down candidate regulators in Nb plants infected with CLso, transmitted by potato psyllids. Here we show the results of one HDPR, BRAP, and one HISU, VAD (Fig. 2). Knocking down BRAP revealed a more susceptible phenotype to CLso (stunted and yellow) (Fig. 2A) and higher bacterial titer in plants (Fig. 2B). Knocking down VAD has the opposite effect, including plants growing much better with lower bacteria titer. We have high confidence that our selected host immune regulators can provide effective protection for citrus because of the similarity to HLB of this pathosystem evaluation. Figure 2. Functional testing of candidate regulators BRAP and VAD with CLso/Nb pathosystem reveals BRAP is a positive regulator and VAD is a negative regulator against CLso infection. A) Two-week-old Nb plants were exposed to CLso positive potato psyllids. Expression knock down of BRAP or VAD regulators used VIGS. Silencing RB gene acts as a control because Nb does not have the RB gene or a closely related homolog (Song et al., 2003). B) The CLso bacteria tier was examined by probe based qPCR in 50ng total genomic DNA of Nb plants shown in panel A. Twelve leaves were collected from four plants from each treatment. Some of the candidate regulators appear to have polymorphisms when comparing HLB-susceptible and HLB-tolerant sequences. Most excitingly, we have identified a novel class of antibacterial peptides that can kill/inhibit CLso growth. We have high confidence in the ability of our candidate regulators to help defend against CLas based on in vivo functional testing with the CLso/Nb system. We have cloned the candidate regulators, and engineering of citrus varieties is planned for immediate execution. The new citrus varieties could be greenhouse tested within 2 years. Citrus therapies would be tested within 1 year. Further functional analysis of these small RNAs and their targets will help identify new regulators in plant disease resistance signaling pathways. At the same time, we established collaborations with other groups on several projects related to small RNA and epigenetics and plant disease resistance. These studies led to several publications. <br><br><b>Publications</b><br>