Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 201 - Plant Genome, Genetics, and Genetic Mechanisms | 999 - Citrus, general/other | 1040 - Molecular biology | 25% |
| 212 - Pathogens and Nematodes Affecting Plants | 999 - Citrus, general/other | 1040 - Molecular biology | 25% |
| 201 - Plant Genome, Genetics, and Genetic Mechanisms | 920 - Orange | 1040 - Molecular biology | 15% |
| 212 - Pathogens and Nematodes Affecting Plants | 920 - Orange | 1040 - Molecular biology | 15% |
| 201 - Plant Genome, Genetics, and Genetic Mechanisms | 930 - Lemon | 1040 - Molecular biology | 10% |
| 212 - Pathogens and Nematodes Affecting Plants | 930 - Lemon | 1040 - Molecular biology | 10% |
Citrus greening/HLB is one of the most devastating diseases of citrus. The mechanism of pathogenesis and possible natural defense responses is hardly known. Therefore, there is an urgent need to understand the mechanisms of natural defense, and to identify important signaling components for crop protection. The output will ultimately help develop resistance crops and reduce the use of toxic fungicide. In addition, HLB, caused by the bacterium Candidatus Liberobacter, is extremely difficult to detect because of the unculturable nature of the bacterium and its low concentration and uneven distribution in the host. Therefore, instead of focusing on the pathogens, we are investigating the defense responses of the host to identify unique host biomarkers for early detection of HLB. The rapid and specific nature of the induction of small RNAs in the host upon pathogen attack makes them potentially ideal biomarkers for early diagnosis. The goal of this project is to 1) identify small RNAs and genes that are important for host defense by comparison analysis between tolerant and susceptible varieties, which will help us to understand the natural defense mechanism as well as the cause of pathogenesis; and 2) find the specific pathogen-induced small RNAs, which can be developed into early diagnosis markers.
Objective I. Identify specific small RNAs from tolerant and susceptible citrus varieties by high throughput profiling We will use Illumina high throughput deep sequencing (UCR Core facility) to profile small RNA libraries. We will choose two tolerant genotypes and two sensitive genotypes for this study. We chose to use Carrizo citrange (X Citroncirus webberi J. Ingram & H. E. Moore) and Eureka lemon (C. limonia Osbeck) as tolerant genotypes because they are not only display little or no symptoms, but also accumulate less Ca. L. bacteria . We chose Valencia sweet orange (C. sinensis (L.) Osbeck) and Cleopatra mandarin (C. reticulata Blanco) as sensitive genotypes beause they not only show very strong symptom of chlorosis and leaf death, but also accumulate high level of bacterium Ca. L.. The small RNA and mRNA libraries are listed as following: Genotypes Early time point 1 Early time point 2 Tolerant Small RNA and mRNA libraries Small RNA and mRNA libraries Carrizo citrange sRNA/mRNA sRNA/mRNA Eureka lemon sRNA/mRNA sRNA/mRNA Sensitive Valencia sweet orange sRNA/mRNA sRNA/mRNA Cleopatra mandarin sRNA/mRNA sRNA/mRNA Total 8 sRNA and 8 mRNA Objective II. Validate these specific small RNAs by Northern blot analysis and in comparison with Spiroplasma-infected conditions. To validate their expression, small RNA will be isolated and subjected to Northern blot analysis. Sequences that give discrete bands at sizes around 21-24 nt which show increased or decreased intensity in infected compared to uninfected plants, will be considered as pathogen-regulated small RNAs. In order to test their specificity, we will examine the presence of these selected small RNAs in other pathogen infected plants to characterize their specificity. The plants that infected by Spiroplasma that induces citrus stubborn diseases and by CTV will be used for specificity analysis. Vidalakis lab will provide those material. Objective III. Identify the targets of these small RNAs by mRNA high throughput RNA-seq to understand their functions in HLB pathogenesis and natural defense responses Small RNAs and their potential targets are likely to be negatively correlated. mRNA profiling help identify and validate these targets. Similarly, HLB-regulated small RNAs and mRNAs may help identify important components in pathogenicity of the greening. We will use Illumina high-throughput deep-sequencing HiSeq to profile the small RNA and mRNA populations of these two distinct tolerant and sensitive varieties. Our lab has extensive experience in deep sequencing and sequencing analysis, which is evidenced by our publications on genome-wide analysis of small RNAs from Arabidopsis and Rice (Padmanabhan and Jin, 2009; Zhou et al., 2009; Chellappan et al., 2010; Zhang et al., 2010). Collaboration has been established between my lab and Dr. Svetlana Folimonova at University of Florida for this project. She will provide the HLB-infected and uninfected tolerant and susceptible citrus plants for our expression profiling of small RNAs and mRNAs.
Target Audience
The PI made every effort to reach to the scientific communities at every levels, including researchers, and students, industry representative, growers, and agricultural officials. The PI has also been actively involved in outreach activities. The PI has talked to the citrus growers in a regular basis, discuss the urgent needs about the HLB control. The PI was also reach to the public, and talked with journalists in press conferences held by the Citrus Research Board, such as presentations at the LAX Airport as a joint effort with the border control to educate the public about the harm of tranporting food from other regions to California.
Changes / Problems
Nothing Reported
Training & Professional Development
This project provided excellent training opportunities to graduate students, postdocs and undergraduate students. They have been trained in the molecular plant pathology field. The graduate students present their work in the Departmental seminar series and the postdoc present their work in sicentific meetings. The postdoc and graduate students also had the opportunity to train the undergraduate students in the lab, who decided to continue their future career in Biology.
Dissemination Streams
During this funding period, the scientific findings were published in highly repected top scientific journals, including Science, Molecular Cell and Nature Communications, etc. Total of 17 research articles were published, and three patent applications were filed. The PI and postdoc have been giving talks in scientific meetings, research institutions every year. The PI also talk with citrus growers multiple times a year. The PI actively participated in the outreach program, such as organizing Career Day for the graduate students and postdocs, giving presentations and discussions during press release events on citrus HLB, etc.
Next Reporting Steps
Nothing Reported