This project aims to develop microRNA (miRNA) knockdown populations in selected crops using our recently developed small tandem target mimic (STTM) technology. This is the first attempt to systematically generate such genetic resources for the plant community, representing a critical step towards comprehensive characterization of miRNA functions. We will further use these mutant populations to investigate the functional conservation and diversification of highly conserved plant miRNAs, and to study the regulatory role of miRNAs in plant-microbe interactions. Hundreds to thousands of miRNAs have been identified from dozens of plant species including agriculturally important crops such as rice, maize, wheat, and soybean. However, their roles in plant development and response against various pathogens and other stresses are largely unknown. The STTM technology is a powerful tool that can robustly trigger the degradation of specific miRNAs in plants, making it possible to dissect miRNA functions. Here, we propose to systemically silence miRNAs in crops by STTM. These miRNA knockdown libraries will be of great use for understanding miRNA functions and will set up genetic basis for crop improvement. Therefore, it is worth making an effort to explore the following specific aims:

Project Objectives:

(1) Generate miRNA mutants and/or STTM constructs that will support research on miRNAs in two monocot (rice and maize) and two dicot (Arabidopsis and soybean) plants.
(2) Examine the roles of miRNAs in plant-microbe interactions using soybean as the host.
(3) Investigate functional conservation and diversification of 6 conserved plant miRNAs by comparing their functions in monocots (rice and maize) and dicots (Arabidopsis and soybean).
(4) Develop digital management resources to support knowledge dissemination, material transfer and outreach.

Experimental Approaches:

1. Generate miRNA mutants and/or STTM constructs in two monocot (rice and maize) and two dicot (Arabidopsis and soybean) plants

         In order generate most effective miRNA STTM constructs we will first systematically analyze miRNAs of plants to be studied using a sequence-clustering tool. Each miRNA family will be targeted by minimal number of STTMs to make sure that all the miRNA family members are targeted for down-regulations. Methods for STTM construction will be based on our published protocol and papers with modifications for effectiveness and construction efficiency. Two types of STTM constructs will be created: (1) Constitutively expressed STTM, and (2) Inducibly/tissue-specifically expressed STTM.
         For production of constitutively expressed transgenic STTM plants, we will generate transgenic STTMs for rice and Arabidopsis as large-scale as possible using well-established plant transformation approaches. For production of transgenic STTM maize and soybean, only six selected miRNAs will be targeted and contracted to Dr. Kan Wang at Iowa State University as a pilot study. Inducibly (estrogen receptor-based XVE system)/tissue-specifically (endosperm specific) expressed STTMs will only be made in constructs for distributions. For the six selected miRNAs, transgenic STTM plants will be generated in homozygous form of sufficient individual lines for functional conservation and diversification analysis.

2. Examine the roles of miRNAs in soybean-microbe interactions using the hairy root system

         To identify those known and unknown miRNAs in soybean-microbe interactions, we will conduct high-throughput small RNA sequencing during different stages of the interaction. STTMs of the identified related miRNAs will be constructed for soybean root transformation. Functions of miRNAs in soybean-pathogen interactions will be investigated in three aspects: 1) soybean-microbe interaction, 2) soybean-nematode interaction, and 3) soybean-rhizobia symbiosis. In order to accurately address miRNA functions in soybean-pathogen interactions, GFP marker-associated STTM will be produced for root transformation. Functions of miRNAs will be evaluated in cells that express GFP.

3. Investigate functional conservation and diversification of 6 conserved plant miRNAs by comparing their functions in monocots (rice and maize) and dicots (Arabidopsis and soybean).

         For functional conservation and diversification of miRNAs we will select 6 miRNAs to generate the constitutively expressed STTM transgenic rice, maize, soybean and Arabidopsis for examination and comparative studies. We will study target genes for the six miRNAs with species-specific developmental impacts. These will include miRNA expression pattern by Northern blot, in situ hybridization, degradome sequencing, RNA-seq and Ribo-seq in transgenic STTMs and their controls.

4. Develop digital management of resources through advanced web database, application, and service for materials transfer and distribution.

         We will develop a web-based tool termed designSTTM to automate STTM vector design. For each specific STTM, designSTTM will automatically store results into the database, and a sketch of a plasmid construct will be plotted with all boundaries between different fragments and lengths of all fragments being clearly labeled. When a design process is completed, an annotated report detailing the steps of STTM construction will be generated. Users can print the sketch of each plasmid vector with the construction protocol.

         We will also develop a web application, materialSTTM, to support the distribution of STTM constructs and transgenic lines. This web application will display the availability of both STTM constructs and transgenic crops lines in which the targeted miRNAs are knocked down. materialSTTM serves as a venue through which homozygous transgenic seeds of each STTM transgene can be distributed to the research community. The web-application of materialSTTM will provide detailed phenotypic description of all homozygous transgenic lines with the images from various developmental stages.

         Finally, we will develop a genome browser for RNA-seq and Ribo-seq of wild-type and STTM mutants. This browser will provide information on global translation status and miRNA-directed translation inhibition, which is currently lacking in plants. We will make them available to the research community through our web portal.

         Technological approaches for the above objective include the standard Linux server, the open-source database server MySQL and the Apache web server. MySQL will be used as the back-end database and Apache will be integrated to create HTML pages to deliver requested web contents back to the users’ browsers. The web application will use PHP for server-side business logic, HTML for content presentation, cascading style sheets (CSS) for formatting, and JavaScript for dynamic client-side behavior.


Information/Materials to be Generated:

         (1) Constructs for constitutively expressed STTMs in rice, maize, soybean, and Arabidopsis;

         (2) Constructs for induciably/tissue-specifically expressed STTMs in rice, maize, soybean, and Arabidopsis;

         (3) Six conserved constitutively expressed STTM transgenic plants of rice, maize, soybean, and Arabidopsis;

         (4) Additional transgenic constitutively expressed STTM plants of rice and Arabidopsis plants for distributions;

         (5) STTM technology training for students selected nationally via a specific STTM workshop held at Michigan Tech;

         (6) Publications on large-scale production of STTM in Arabidopsis and rice, as well as role of miRNAs in functional conservation and diversification among different plant species and in plant-pathogen interactions.

Participants :

Guiliang Tang, Michigan Technological University
Xuemei Chen, UC Riverside;
Wenbo Ma, UC Riverside;
Harold Trick, Kansas State University;
Hairong Wei, Michigan Technological University