INVESTIGATION OF PLANT DISEASE RESISTANCE SIGNALING AND INNATE IMMUNITY

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0207229
• Project Start Date: Oct 1, 2006
• Project End Date: Sep 30, 2011
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF CALIFORNIA, RIVERSIDE
(N/A)
RIVERSIDE,CA 92521

Performing Department
Plant Pathology, Riverside

Non Technical Summary
Plant diseases caused by various pathogens can lead to huge loss in crop productivity each year. For example, late blight, one of the most devastating plant diseases, causes worldwide losses in potato and tomato are estimated to exceed $3 billion every year (Fry, 1997); sudden oak death, caused by a different species within the same genus Phytophthora, has become a major problem in the forest industry in the States, especially oak woodland in California and western region. Although agrochemicals remain the major tool for plant disease control, they can be costly and toxic to the environment and human health. In addition, many pests and pathogens can quickly develop resistance to these chemicals, negating their utility. Therefore, more effective and sustainable strategies for managing plant diseases are needed. One of the attractive strategies for plant disease control is to utilize plant natural defense system, so-called plant innate immunity (Asai et al., 2002; Hammond-Kosack and Parker, 2003). However, the molecular mechanisms of plant disease resistance are still poorly understood. The goal of my research program is to identify key elements of these defense systems, which can then be used to develop effective and more sustainable strategies for disease control. We will use molecular functional genomics approaches to study late blight resistance signaling and molecular mechanism of sudden oak death.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	1310	1040	10%
201	1460	1040	10%
201	2300	1040	10%
212	1310	1040	40%
212	1460	1040	30%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1310 - Potato; 1460 - Tomato; 2300 - Weeds;

Field Of Science
1040 - Molecular biology;

Keywords

late blight resistance

sudden oak death

plant innate immunity

Goals / Objectives
The research goal in my lab is to identify key signaling components in plant disease resistance and to understand the regulatory mechanism of disease resistance signaling and plant innate immunity. This study will ultimately help develop effective means of disease control in the fields. The signal transduction of late blight disease resistance and the molecular mechanism of sudden oak death will be our major focus in this study. Although plant breeders have been searching a long time for a source of genetic resistance to P. infestans, little progress has been made. Recently, a broad-spectrum late blight resistance gene (RB) has been identified from wild potato species, which confers resistance to all known races of the late blight pathogen (Song et al., 2003; van der Vossen et al., 2003). Its signal transduction pathway is totally unknown. Understand this RB-mediated late blight resistance has obvious and considerable economic importance. Sudden oak death, caused by P. ramorum, continues to spread locally and regionally to over 20 states, which urges scientists to study the mechanisms and to develop new treatment strategies. However, conduct genetic and molecular studies in woody plants is extremely challenge. Since plant innate immunity and its signaling pathways are evolutionarily conserved (Meyers BC, 2005), establish a model system for plant-P. ramorum interaction becomes a desirable approach for this study. We have recently identified different tomato species that are either susceptible or resistance to P. ramorum, which allow us to characterize the molecular mechanisms of plant resistance and susceptibility to this pathogen. The following objectives have been proposed for the next five years: Objective 1. Use virus-induced gene silencing (VIGS) to study the role of known disease resistance signaling components in RB-mediated resistance Objective 2. Perform global expression analysis of inoculated and un-inoculated RB-transgenic potato Objective 3. Conduct global expression profiling analysis on susceptible/resistance tomato before and after P. ramorum infection. Objective 4. Compare the expression profiling results with Arabidopsis data and examine the functions of common genes in Arabidopsis Objective 5. Examine the functions of newly identified genes by VIGS

Project Methods
To achieve our goal, my research plan combines investigation of model systems as well as solanaceous crops. Utilization of the model system (Arabidopsis) will allow relatively rapid advances in our understanding of the molecular basis for plant defense. This knowledge will then facilitate the identification of the key signal transduction components of late blight disease resistance in potato. We will use global gene expression profiling by microarray hybridization and comparison analysis to identify common signaling components that are required for shared innate immunity pathways. Then we will use powerful functional genetic tool---virus-induced gene silencing (VIGS) to assess the function of identified candidate genes in RB signaling pathway in potato and potential P. ramorum resistance in tomato. These studies will generate large amount of information for future studies. It will help develop effective disease control methods that are environmental-benign and long-lasting, and ultimately allow breeders and farmers to grow economically important crops with improved pathogen resistance traits.

Progress 10/01/06 to 09/30/11

Outputs
Target Audience: The PI made every effort to reach to the scientific groups, including researchers, postdoc fellows and students in the field of Plant-Microbial interaction through sicentific meeting presentations, publications, meeting reports, and outreach activities. The PI also talked to the citrus growers in a regular basis, discuss the urgent needs about the HLB control. The PI was invitied by the Citrus Research Board to give presentations at the Press Conference for CItrus HLB disease in Walnut City, California. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project provided excellent training opportunities to graduate students, postdocs and undergraduate students. 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 career in Biology. How have the results been disseminated to communities of interest? The resuts of this project has been disseminated to scientific communities and interest parties. The activity includes: publish research articles in highly respected journals. One patent application was filed. The PI and postdoc have been giving talks at scientific meetings or to citrus growers multiple times a year. The PI also actively took part in the outreach program by giving presentations during outreach activities on citrus HLB topics, etc. What do you plan to do during the next reporting period to accomplish the goals? This funding period set a strong foundation for this research program, and the deep sequencing results provided many projects for future followup in-depth functional analysis, which will lead to the findings of new regulators and players in the plant defense network.

Impacts
What was accomplished under these goals? We have made significant progress during the entire project period, and established a solid program in the field of small RNAs and epigenetics in plant-microbial interaction. Over the last 5 years, we published total of 17 research papers, and 3 book chapters. The major achievement is listed below: 1. Identification of host small RNAs for early diagnosis of Citrus Greening disease and Citrus Stubborn disease. “Huanglongbing” (HLB, citrus greening) and Citrus Stubborn are two serious diseases of citrus plants and citrus industry is one of the most important industries in California. The early diagnosis of these diseases is critical. 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. Similarly, the available diagnostic protocols for citrus stubborn disease, which targeting the pathogen Spiroplasma citri, are often unreliable. 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 and Stubborn, in collaboration with Dr. Bowman from USDA, FL and Dr. Vidalakis at UCR. 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. Our recent deep-sequencing experiments have identified a group of conserved citrus miRNAs and numerous siRNAs. Some of them are rapidly induced by HLB-infection. These sRNAs could be developed into early diagnostic markers for HLB. 2. Genome-wide analysis of pathogen-regulated endogenous small RNAs in plant defense responses in Arabidopsis. Using high throughput sequencing, our lab has identified a group of pathogen-regulated endogenous small RNAs (including many new miRNAs and siRNAs) that are regulated by the infection of bacterial (Pseudomonas syringae) and fungal (Alternaria brassicicola and Botrytis cinerea) pathogens. Identification of many more pathogen-regulated siRNAs has laid the foundation for my long term research program. The following are some examples of the output: A. We identified the first plant endogenous siRNA contributes to resistance gene RPS2-mediated race-specific disease resistance by repressing a putative negative regulator of the resistance pathway. This work was published in PNAS. B. In addition to typical 20-25nt miRNAs and siRNAs, we also discovered a novel class of endogenous small RNAs, referred to as long siRNAs (lsiRNAs). These lsiRNAs are 30–40 nt in length and are induced by bacterial infection or specific growth conditions. One lsiRNA is induced in plants specifically by P. syringae carrying the effector avrRpt2, and down-regulates a putative RNA binding protein gene, possibly through a novel mode of action. This work was published in Genes & Development, and was highlighted by Nature and selected by Faculty of 1000. C. We discovered that Arabidopsis RNA silencing pathway component Argonaute 2 (AGO2) is highly induced by the bacterial pathogen Pseudomonas syringae pv. tomato (Pst), and positively regulates antibacterial immunity. This work was published in Molecular Cell 2011. D. In the large datasets of small RNAs generated by deep sequencing, we have identified a group of siRNAs that are generated from natural antisense transcript (NAT) regions (BMC Molecular Biology, 2008, Pacific Symposium on Biocomputing, 2008). We also found that some miRNA genes could give rise to more than one functional miRNA. These miRNA-sibling RNAs are potentially functional and exist broadly in the plant kingdom, including rice, moss, Medicago and Populus. This work was published in Genome Biology 2010. We also found that some MIR genes could give rise to both miRNAs and siRNAs at the same site and cooperatively silence their targets at both the posttranscriptional level and transcriptional level. We also showed that this type of dual-functional MIR gene exists broadly in plants. This work was published in Nucleic Acid Research 2010. 4. The other project was to dissect the signaling pathway of resistance gene RB-mediated resistance to Phytophthora infestans, in collaboration with Dr. Coffey from UCR and Dr. Jiang from University of Wisconsin. Using VIGS, we identified a group of MAP kinases and other signaling components that play a role in RB-mediated resistance.

Publications

• Type: Journal Articles Status: Published Year Published: 2006 Citation: 1. Xiaoming Zhang, Hongwei Zhao, Shang Gao, Wei-Chi Wang, Surekha Katiyar-Agarwal, Hsien-Da Huang, Natasha Raikhel and Hailing Jin*: Arabidopsis Argonaute 2 regulates innate immunity via miRNA393*-mediated silencing of a Golgi-localized SNARE gene MEMB12. Molecular Cell 2011, 42, 356-366. 2. Panagiotis F. Sarris, Shang Gao, Konstantinos Karademiris, Hailing Jin, Kriton Kalantidis and Nickolas J. Panopoulos: Phytobacterial Type III Effectors HopX1, HopAB1 and HopF2 Enhance Sense-Post Transcriptional Gene Silencing Independently of Plant R Gene-Effector Recognition. Mol Plant Microbe Interact. 2011, DOI: 10.1094/MPMI-01-11-0010. 3. Weixiong Zhang*, Shang Gao, Xiang Zhou, Padmanabhan Chellappan, Zheng Chen, Xuefeng, Zhou, Xiaoming Zhang, Nyssa Fromuth, Gabriela Coutino, Michael Coffey, and Hailing Jin*: Bacterial-responsive microRNAs regulate plant innate immunity by modulating plant hormone networks. Plant Molecular Biology 2011, 75, 93-105. 4. Niu DD, Liu HX, Jiang CH, Wang YP, Wang QY, Jin H, Guo J. The Plant Growth-Promoting Rhizobacterium Bacillus cereus AR156 Induces Systemic Resistance in Arabidopsis thaliana by Simultaneously Activating Salicylate- and Jasmonate/Ethylene-dependent Signaling Pathways. Mol Plant Microbe Interact. 2011, 24, 533-42. 5. Zhang W*, Gao S, Zhou X, Xia J, Chellappan P, Zhou X, Zhang X, Jin H.*: Multiple distinct small RNAs originated from the same microRNA precursors. Genome Biol. 2010, 11, R81. 6. Padmanabhan Chellappan, Jing Xia, Xuefeng Zhou, Shang Gao, Xiaoming Zhang, Gabriela Coutino, Franck Vazquez, Weixiong Zhang and Hailing Jin*: siRNAs from miRNA sites mediate DNA methylation of target genes. Nucleic Acid Research, 2010, 38, 6883-94. 7. Surekha Katiyar-Agarwal and Hailing Jin*: Role of Small RNAs in host-microbe interactions. Annu. Rev. Phytopathol. 2010, 48, 225-246. 8. He XJ, Hsu YF, Zhu S, Wierzbicki AT, Pontes O, Pikaard CS, Liu HL, Wang CS, Jin H, Zhu JK. An effector of RNA-directed DNA methylation in arabidopsis is an ARGONAUTE 4- and RNA-binding protein. Cell 2009,137, 498-508. 9. Ni Li, Carolyn Jablonowski, Hailing Jin and Wenwan Zhong: Stand-Alone Rolling Circle Amplification Combined with Capillary Electrophoresis for Specific Detection of Small RNA. Analytical Chemistry 2009. 81, 4906-13. 10. Padmanabhan Chellappan, Xiaoming Zhang and Hailing Jin*: Host small RNAs are big contributors in plant innate immunity. Current Opinion in Plant Biology, 2009, 12, 465-72. 11. Xuefeng Zhou, Ramanjulu Sunkar, Hailing Jin, Jian-Kang Zhu and Weixiong Zhang: Genome-wide identification and analysis of small RNAs originated from natural antisense transcripts in Oryza sativa. Genome Research, 2009, 19, 70-78. 12. Hailing Jin* (co-corresponding author), Vladimir Vacic, Thomas Girke, Stefano Lonardi and Jian-Kang Zhu*: Small RNAs and the regulation of cis-natural antisense transcripts in Arabidopsis. BMC Molecular Biology, 2008, 9:6. 13. Hailing Jin*: Endogenous Small RNAs And Antibacterial Immunity in Plants. FEBS Letters, 2008, 582, 2679-84. 14. Surekha Katiyar-Agarwal, Shang Gao, Adam Vivian-Smith and Hailing Jin*: A novel class of bacteria-induced small RNAs in Arabidopsis. Genes & Development 2007, 21, 3123-3134. 15. Vladimir Vacic, Hailing Jin, Jian-Kang Zhu and Stefano Lonard: A probabilistic method for small RNA flowgram matching. Pacific Symposium on Biocomputing, 2008, 13, 75-86. 16. Katiyar-Agarwal S, Morgan R, Dahlbeck D, Borsani O, Villegas A Jr, Zhu JK, Staskawicz B*, Jin H.* 2006. A pathogen-inducible endogenous siRNA in plant immunity. Proc. Natl. Acad. Sci. USA 103: 1802-18007. 17. Hailing Jin*, Songtao Li and Andy Villegas Jr.: Down Regulation of 26S Proteasome Subunit RPN9 Inhibits Viral Systemic Transport and Alters Plant Vascular Development. Plant Physiology, 2006, 142, 651-661.

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: We have made good progress during the last year. We did in depth analysis on the small RNA deep sequencing data from bacteria and fungal-challenged Arabidopsis. We also identified a good miRNA that are specifically induced by HLB and can be developed into a potential early diagnosis marker. The major findings are outlined below. 1. We have identified one miRNAs and two siRNAs from citrus plants that are specifically induced by Candidatus Liberibacter from both deep sequencing results as well as Northern blot analysis. Subsequent analysis showed that these small RNAs couldn't be detected from Spiroplasma-infected plants, suggesting that they are rather specific. The specificity and the early induction nature made them good candidates for early diagnosis markers. We are in the process of patenting them. 2. We identified more than 17K and 56K unique nat-siRNAs from stress-challenged Arabidopsis and rice, respectively. These nat-siRNAs were enriched in the overlapping regions of NATs and exhibited either site-specific or distributed patterns with strand bias. Our analysis significantly advances our understanding of the biogenesis and function of nat-siRNAs in plants. 3. In the small RNA biogenesis pathway, we have discovered that Arabidopsis AGO2 is highly induced by the bacterial pathogen Pseudomonas syringae pv. tomato (Pst). Genetic analysis demonstrated that AGO2 functions in antibacterial immunity. We further demonstrated that AGO2 functions in antibacterial immunity by binding miR393b* to modulate exocytosis of antimicrobial PR proteins. We have already submitted this manuscript. 4. We continued working on a novel class of dual functional miRNA MIR genes that give rise to two small RNA species at the same loci, a 21-nt species and a 24- or 30-nt species. We found that the 21-nt small RNAs are typical miRNAs whereas the 24-nt and 30-nt small RNAs are siRNAs. We further demonstrate that these MIR gene-derived siRNAs and long siRNAs (lsiRNAs) can direct DNA methylation at some of their target loci in trans. This work was published in Nucleic Acid Research in 2010. 5. We also finished the study on miRNA genes that yield multiple distinct miRNA-like RNAs in addition to miRNAs or miRNA*s. We refer to these as miRNA-sibling RNAs (msRNAs). Our systematic examination of publicly available high-throughput small-RNA deep sequencing data for rice, moss, Medicago and Populus shows that msRNAs exist broadly in the plant kingdom. This work was published in Genome Biology in 2010. 6. We have also identified a group of bacterial-regulated miRNAs targets protein-coding genes that are involved in plant hormone signaling, including those in auxin, ABA, and JA pathways. This work was published in Plant Molecular Biology earlier this year. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
During the study of pathogen-induced small RNAs and small RNA pathway components in Arabidopsis, we have identified many new siRNAs and miRNAs that are regulated by pathogen infections, which may play an important role in plant defense. In depth analysis of several small RNAs and their targets led us to the identification of several important regulators for plant immunity. We have discovered 28 citrus miRNAs that are conserved within Arabidopsis and citrus, as well as over one dozen of new miRNAs. We identified HLB-induced smallRNAs that could potentially be developed to early diagnosis markers. In addition, further investigation of one of the HLB-induced small RNA revealed one of the major causes for HLB pathogenesis, which may lead to effective means for HLB control. 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.

Publications

• 4. Padmanabhan Chellappan, Jing Xia, Xuefeng Zhou, Shang Gao, Xiaoming Zhang, Gabriela Coutino, Franck Vazquez, Weixiong Zhang and Hailing Jin: siRNAs from miRNA sites mediate DNA methylation of target genes. Nucleic Acid Research, 2010, doi:10.1093/nar/gkq590.
• 1. Weixiong Zhang, Shang Gao, Xiang Zhou, Padmanabhan Chellappan, Zheng Chen, Xuefeng, Zhou, Xiaoming Zhang, Nyssa Fromuth, Gabriela Coutino, Michael Coffey, and Hailing Jin: Bacterial-responsive microRNAs regulate plant innate immunity by modulating plant hormone networks. Plant Molecular Biology 2011, 75(1-2):93-105.
• 2. Niu DD, Liu HX, Jiang CH, Wang YP, Wang QY, Jin H, Guo J. The Plant Growth-Promoting Rhizobacterium Bacillus cereus AR156 Induces Systemic Resistance in Arabidopsis thaliana by Simultaneously Activating Salicylate- and Jasmonate/Ethylene-dependent Signaling Pathways. Mol Plant Microbe Interact. 2011 Jan 3. [Eput ahead of print].
• 5. Hailing Jin & Jian-Kang Zhu: A viral suppressor protein inhibits host RNA silencing by hooking up with Argonautes. Genes & Development, 2010, 24, 853-856.
• 6. Hailing Jin & Jian-Kang Zhu: How many ways are there to generate small RNAs Molecular Cell, 2010, 38(6):775-7.
• 7. Surekha Katiyar-Agarwal and Hailing Jin: Role of Small RNAs in host-microbe interactions. Annu. Rev. Phytopathol. 2010. 48:225-246.
• 3. Zhang W, Gao S, Zhou X, Xia J, Chellappan P, Zhou X, Zhang X, Jin H.: Multiple distinct small RNAs originated from the same microRNA precursors. Genome Biol. 2010 Aug 9;11(8):R81.
• 8. Zhihuan Gao, Hai-Liang Liu, Lucia Daxinger, Olga Pontes, Xinjian He, Weiqiang Qian, Mingtang Xie, Zdravko J. Lorkovic, Shoudong Zhang, Dominique Pontier, Thierry Lagrange, Steven E. Jacobsen, Hailing Jin, Antonius J.M. Matzke, Marjori Matzke, Craig S. Pikaard, and Jian-Kang Zhu: An RNA polymerase II- and AGO4-associated protein acts in RNA-directed DNA methylation. Nature, 2010, 465, 106-110.
• 9. Agee AE, Surpin M, Sohn EJ, Girke T, Rosado A, Kram BW, Carter C, Wentzell AM, Kliebenstein DJ, Jin HC, Park OK, Jin H, Hicks GR, Raikhel NV: MODIFIED VACUOLE PHENOTYPE1 is an Arabidopsis myrosinase-associated protein involved in endomembrane protein trafficking. Plant Physiol., 2010, 152 (1):120-32.

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: The goal of my research is to identify early diagnosis markers for serious plant diseases and to develop effective means for disease control by utilizing plant natural defense systems. RNA silencing, mediated by small interfering RNAs (siRNAs), has evolved as an antiviral defense mechanism in plants and animals. However, It was not known whether host cells also use siRNAs as an antibacterial defense mechanism in eukaryotes. My lab has been focusing on pathogen-induced small RNAs in several plant-microbial interaction systems, including citrus greening (HLB) and citrus stubborn ---citruses, bacterial and fungal pathogens --- Arabidopsis, and P. infestans --- potato. Citrus greening or "Huanglongbing" (HLB), caused by bacteria Candidatus Liberobacter, is one of the most devastating diseases of citrus. The serious spread of HLB in Florida is of great concerns to citrus industry as well as scientists. Recent detection of HLB carrying Psyllids in Texas makes it an urgent need for scientist to develop effective measures to prevent its further spread. To prevent its further spread, early diagnosis before the appearance of the dreadful symptoms is particularly important. However, the unculturable nature of the bacteria and their low concentration and uneven distribution in the hosts make it extremely difficult to detect HLB infection. Instead of focusing on the bacteria, we took advantage of host rapid defense responses and propose to identify unique host biomarkers for early diagnosis of HLB. Some host small RNAs are rapidly and specifically induced by pathogens, which makes them one of the most attractive markers for early diagnosis. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
During the study of pathogen-induced small RNAs, we have identified a group of small RNAs that are induced by pathogen infections, which may play an important role in plant defense. We have generated 4 million small RNA reads from citrus-HLB libraries and discovered 28 citrus miRNAs that are conserved within Arabidopsis and citrus, as well as over one dozen of new miRNAs. We identified HLB-induced siRNAs that could potentially be developed to early diagnosis markers. 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.

Publications

• 1.Xuefeng Zhou, Ramanjulu Sunkar, Hailing Jin, Jian-Kang Zhu and Weixiong Zhang: Small RNAs generated from natural antisense transcripts in Oryza sativa. Genome Research, 2009, 19, 70-78. 2.He XJ, Hsu YF, Zhu S, Wierzbicki AT, Pontes O, Pikaard CS, Liu HL, Wang CS, Jin H, Zhu JK. An Arabidopsis homolog of the transcription elongation factor SPT5 is a novel RNA-binding protein that interacts with ARGONAUTE 4 and is required for RNA-directed DNA methylation. Cell 2009,137 (3):498-508. 3.Hailing Jin* (co-corresponding author), Vladimir Vacic, Thomas Girke, Stefano Lonardi and Jian-Kang Zhu: Small RNAs and the regulation of cis-natural antisense transcripts in Arabidopsis. BMC Molecular Biology, 2008, 9:6. doi:10.1186/1471-2199-9-6. 4.Agee AE, Surpin M, Sohn EJ, Girke T, Rosado A, Kram BW, Carter C, Wentzell AM, Kliebenstein DJ, Jin HC, Park OK, Jin H, Hicks GR, Raikhel NV: MODIFIED VACUOLE PHENOTYPE1 is an Arabidopsis myrosinase-associated protein involved in endomembrane protein trafficking. Plant Physiol., 2010, 152(1):120-32. 5.Atsunari Tsuchisaka, Guixia Yu, Hailing Jin, Jose M. Alonso, Joseph R. Ecker, Xiaoming Zhang, Shang Gao and Athanasios Theologis: A Combinatorial Interplay Among the 1-Aminocyclopropane-1-carboxylate Isoforms Regulates Ethylene Biosynthesis in Arabidopsis thaliana. Genetics, 2009, 183(3):979-1003. 6.Padmanabhan Chellappan and Hailing Jin*: Discovery of plant microRNAs and short-interfering RNAs by deep parallel sequencing. Methods in Molecular Biology, 2009, 495, 121-132. 7.Iida K, Jin H, Zhu, JK. Bioinformatics analysis suggests base modifications of tRNAs and miRNAs in Arabidopsis thaliana. BMC Genomics 2009, 10:155. 8.Ni Li, Carolyn Jablonowski, Hailing Jin and Wenwan Zhong: Stand-Alone Rolling Circle Amplification Combined with Capillary Electrophoresis for Specific Detection of Small RNA. Analytical Chemistry 2009. DOI: 10.1021/ac900578a.

Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: Outputs The goal of my research is to understand the mechanisms of plant disease resistance and innate immunity and ultimately to develop effective means for disease control by utilizing plant natural defense systems and to identify early diagnosis markers for serious plant diseases. RNA silencing, mediated by small interfering RNAs (siRNAs), has emerged to be imporant for plant defense mechanism in eukaryotes. My lab has been focusing on pathogen-induced small RNAs in several plant-microbial interaction systems, including bacterial and fungal pathogens-Arabidopsis, citrus-greening (HLB) and citrus stubborn-citruses and P. infestans-potato. Citrus greening or HLB, caused by bacteria Candidatus Liberobacter, is one of the most devastating diseases of citrus. The serious spread of HLB in Florida is of great concerns to citrus industry as well as scientists. Recent detection of HLB carrying Psyllids in Texas makes it an urgent need for scientist to develop effective measures to prevent its further spread. To prevent its further spread, early diagnosis before the appearance of the dreadful symptoms is particularly important. Some host small RNAs are rapidly and specifically induced by pathogens, which makes them one of the most attractive markers for early diagnosis. During this year, we made significant progress in this project, which include: We have isolated and purified small RNAs from HLB-infected plants and HLB-free control plants at 5 weeks and 9 weeks post inoculation. The small RNAs were ligated with 5'- and 3'- RNA adaptors and reverse transcripted into cDNA libraries. Small RNA libraries were deep-sequenced using Illumina SBS sequencer. We obtained total of 4 million reads and did following analysis on the small RNA sequenced and the validation was also initiated. We have also fulfilled the proposed activities on small RNA library construction and sequencing using the high-throughput parallel sequencing on P. infestans-infected potato and Pseudomonas and Botrytis and Alternaria-infected Arabidopsis. We are in the process of analyzing and validating the small RNA datasets. PARTICIPANTS: Hailing Jin PI, oversee the whole project Padmanabhan Chellappan, assistant specialist, made all the small RNA libraries and is in charge of small RNA analysis Hongwei Zhao, postdoc fellow, study the function of small RNA Shang Gao, graduate student, validate the small RNAs. Partner Organizations: Citrus Research Board Collaborators and contacts: Kim Bowman, USDA, FL TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: No major changes have been made in this project

Impacts
For the citrus project, a total of over 4 million reads were obtained. After removing the sequences that match non-sRNAs, such as tRNA, rRNA, snoRNA and snRNA and removing the sequences longer or shorter than expected sizes, we obtained total of 2,656,558 reads. We then assembled all the 483,092 citrus ESTs that are publicly available (included the large dataset of the newly released citrus ESTs) and got 26,153 contigs and 255,108 singlets. We mapped all the small RNA reads onto the citrus contigs and singlets. Excitingly, total of 704,773 small RNAs (excluded the tRNA and rRNA matching reads) can match citrus sequences. We discovered 28 citrus miRNAs that are conserved within Arabidopsis and citrus, which confirmed the success of our small RNA cloning and data analysis. We also found some of the small RNAs are differentially expressed in different libraries. Small RNA validation and cross-hybridization analysis are underway. We have also finished analyzing Pseudomonas and Botrytis and Alternaria-infected Arabidopsis. We found a group of new endogenous siRNAs in Arabidopsis. We are in the process of analyzing and validating the small RNA datasets.

Publications

• Hailing Jin (corresponding author), Vladimir Vacic, Thomas Girke, Stefano Lonardi and Jian-Kang Zhu: Small RNAs and the regulation of cis-natural antisense transcripts in Arabidopsis. BMC Molecular Biology, 2008, doi:10.1186/1471-2199-9-6.
• Vladimir Vacic, Hailing Jin, Jian-Kang Zhu and Stefano Lonard: A probabilistic method for small RNA flowgram matching. Pacific Symposium on Biocomputing, 2008, 13, 75-86.
• Hailing Jin*: Endogenous Small RNAs And Antibacterial Immunity in Plants. FEBS Letters, 2008, 582 (18), 2679-84.
• Wen-Xue Li, Youko Oono, Jianhua Zhu, Xin-Jian He, Jian-Min Wu, Iida Kei, Xiao-Yan Lu, Xinping Cui, Hailing Jin and Jian-Kang Zhu: Transcriptional and posttranscriptional regulation of a transcription factor important for drought resistance in Arabidopsis. Plant Cell, 2008, 10.1105/tpc.108.059444.
• Xuefeng Zhou, Ramanjulu Sunkar, Hailing Jin, Jian-Kang Zhu and Weixiong Zhang: Small RNAs generated from natural antisense transcripts in Oryza sativa. Genome Research, 2008, doi:10.1101/gr.084806.108.
• Padmanabhan Chellappan and Hailing Jin*: Discovery of plant microRNAs and short-interfering RNAs by deep parallel sequencing. Methods in Molecular Biology, 2008, V495, 121-132.

Progress 01/01/07 to 12/31/07

Outputs
Plant diseases caused by various pathogens can lead to huge loss of crop productivity each year. The major method farmers are using for disease control is still the extensive use of agrochemicals, which are not only costly but also toxic to the environment and human health. The goal of my research is to understand the mechanisms of plant disease resistance and innate immunity and ultimately to develop effective means for disease control by utilizing plant natural defense systems. My lab has been focusing on pathogen-induced small RNAs in several plant-microbial interaction systems, including bacterial and fungal pathogens-Arabidopsis, citrus-greening (HLB) and citrus stubborn-citruses and P. infestans-potato. RNA silencing, mediated by small interfering RNAs (siRNAs), has evolved as an antiviral defense mechanism in plants and animals. However, It was not known whether host cells also use siRNAs as an antibacterial defense mechanism in eukaryotes. My lab discovered the first plant endogenous siRNA that is specifically induced by the bacterial pathogen Pseudomonas syringae. Small RNA library sequencing using the high-throughput parallel sequencing allows us to identify many new pathogen-induced small RNAs. We are in the process of constructing, sequencing and analyzing small RNA libraries made from HLB-infected citrus leaves, P. infestans-infected potato and Pseudomonas and Botrytis and Alternaria-infected Arabidopsis.

Impacts
We identified the first plant endogenous siRNA contributes to resistance gene RPS2-mediated race-specific disease resistance by repressing a putative negative regulator of the resistance pathway. This work was published in PNAS. During the study of pathogen-induced small RNAs, we recently identified a novel class of small RNAs, long siRNAs (lsiRNAs), which are 30-40 nt and share many common features with known siRNAs. The lsiRNAs identified so far are induced by pathogen infection or under specific growth conditions. One of the lsiRNAs, AtlsiRNA-1, is generated from SRRLK/AtRAP NAT pair and specifically induced by the bacterium Pseudomonas syringae carrying effector avrRpt2. Induction of AtlsiRNA-1 silences AtRAP, which encodes a RAP-domain protein involved in disease resistance. We further demonstrated that the mode of action of AtlsiRNA-1 is to destabilize the target mRNA by decapping and 5'-3' degradation. This work has been published in Genes & Development in Nov 2007 and was chosen by Nature for its Research Highlights in Dec 2007. We have also analyzed all the available small RNAs from public databases and our own sequencing results, and identified many small RNAs that target natural antisense transcript (NAT) regions in the genome. Together with the expression analysis of the NATs, we conclude that small RNA-mediated gene silencing may serve as one of the gene expression regulatory mechanisms for at least a subgroup of NATs in Arabidopsis. This work was published in BMC Molecular Biology, Jan 2008. The related methodology on small RNA sequencing analysis was published in Pacific Symposium on Biocomputing, Jan 2008.

Publications

• Surekha Katiyar-Agarwal, Shang Gao, Adam Vivian-Smith and Hailing Jin: A novel class of bacteria-induced small RNAs in Arabidopsis. Genes & Dev., 2007, 21, 3123-3134. (This paper was selected by Nature in its research highlights of December 2007)
• Surekha Katiyar-Agarwal and Hailing Jin: Discovery of Pathogen-Regulated Small RNAs in Plants. Methods in Enzmology, 2007, 215-227.
• Vladimir Vacic, Hailing Jin, Jian-Kang Zhu and Stefano Lonard: A probabilistic method for small RNA flowgram matching. Acific Symposium on Biocomputing, 2008, 13, 75-86.
• Surekha Katiyar-Agarwal, Rebekah Morgan, Douglas Dahlbeck, Omar Borsani, Andy Villegas Jr., Jian-Kang Zhu, Brian Staskawicz and Hailing Jin: A pathogen-inducible endogenous siRNAs in plant immunity. PNAS 2006, 103, 18002-18007. (This paper was selected by Faculty of 1000 Biology and was also highlighted in the In this issue of PNAS)
• Hailing Jin* (co-corresponding author), Vladimir Vacic, Thomas Girke, Stefano Lonardi and Jian-Kang Zhu: Small RNAs and the regulation of cis-natural antisense transcripts in Arabidopsis. BMC Molecular Biology, 2008, 14, doi:10.1186/1471-2199-9-6.