Progress 06/01/23 to 05/31/24
Outputs
Target Audience:The target audience of this project includes scientists in the field of agricultural sciences, growers, agricultural professionals and other stakeholders. In addition, we will also disseminate the findings to undergraduate and graduate students through lectures and labs in the College of Agriculture at Purdue University. The Purdue Nematology Lab website will serve as the hub of posting project-related information (https://ag.purdue.edu/department/btny/labs/zhang/index.html). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided systemic training to a postdoctoral researcher (Chenmi Mo). The training comprised improvements in molecular and nematology techniques, such as Gateway cloning, tomato root transformation, RKN infection and phenotyping, single cell RNA-seq library construction and also in computational data analysis of mRNA-seq, sneRNA-seq and sRNA-seq. The postdoc will give a poster presentation at the Annual Conference of Society of Nematologists in August 2024. In addition, the project trained three undergraduate students, Marilyn Vargas (Biochemistry major), Olivia Kelley (Biological Science major) and Ami Propst-Zuverza (Plant Science major). Both Marilyn and Olivia are first-generation of college students at Purdue University. The undergraduate students were trained for nematode extraction, inoculation of tomato plants, counting galls formed on roots, extracting and enumerating different development stages of RKN. The experience provided them with opportunities to design and conduct experiments, collect and analyze the data under supervision at the beginning two months and then more independently afterwards. How have the results been disseminated to communities of interest?The main avenue of results dissemination so far has been a presentation during the Faculty Update (Lei Zhang) of the Department of Botany and Plant Pathology at Purdue University on April 24, 2024. What do you plan to do during the next reporting period to accomplish the goals?For Objective 1, we will continue screening at least 50 more candidate protein-coding genes based on the newly generated sneRNA-seq data on the early development stages of M. incognita. The sex-related genes identified will be critical for further studying the molecular mechanism of RKN sex determination and development. These genes are promising targets to develop new tactics of RKN control by disrupting female development or promoting the sex development to males. For the Objective 2, we will select ~ 60 candidate miRNA genes and test their function in RKN sex development using the host-derived artificial miRNA (amiRNA) strategy during the 2nd-year of the project.
Impacts
What was accomplished under these goals?
Root-knot nematodes (RKN; Meloidogyne spp.) are among the critical yield-limiting pests of specialty crop production in the US and around the world. The presence and damage caused by RKN in vegetable production spread across tropical, subtropical, and temperate regions. Effective RKN management methods are critical needs for vegetable growers to achieve successful and profitable production. The long-term goal of research is to develop sustainable methods to reduce RKN populations by interfering with nematode development and reproduction. The aim of this project is to identify genes of M. incognita important for the nematode sex development. The RKN genes critical for nematode sex development and reproduction are promising to be explored as targets to develop tactics for nematode management. The Objective 1 is to identify nematode protein-coding genes involved in M. incognita sex development. We have completed a comprehensive analysis of the low-input RNA-seq data on the three M. incognita developmental stages: pre-parasitic 2nd-stage juveniles (pre-J2s), adult females and males. More than 100 candidate genes have been selected based on gene expression patterns and GO enrichment analysis. In order to identify genes important for RKN sex development, host-induced gene silencing (HIGS) deployed through Agrobacterium rhizogenes-mediated tomato root transformation was used to express dsRNA targeting specific candidate genes. For each candidate gene selected, closely related orthologs were also considered when designing primers for dsRNA. So far, we have screened 51 candidate genes using the HIGS approach, and we have successfully identified and confirmed by repeating the experiments two genes, knocked down of which leading to the altered ratio of adult females and males on tomato roots, indicating that these genes are important for M. incognita sex development. The expression patterns of the two genes at early developmental stages were also investigated and showed different expression patterns between two sexes compared to pre-J2s. Since it is relatively difficult to collect enough RKN samples of early developmental stages for low-input RNA-seq, we adapted and optimized a Single-NEmatode RNA-seq (sneRNA-seq) which allowed us to study the global gene expression of different sexes at early developmental stages. We are currently analyzing the sneRNA-seq data to identify more candidate genes to be introduced in the HIGS screening pipeline in the 2nd year of the project. The Objective 2 is to conduct expression profiling and characterize microRNA (miRNA) genes critical for M. incognita sex development. As a first step, we have collected samples of three developmental stages of M. incognita: pre-J2, adult female and male, with four biological replicates for each developmental stage. Total RNAs including small RNAs (17-200 nt) were extracted from each sample. Total RNAs were sent to Real Seq Biosciences (CA, USA) for purification of small RNAs and small RNA-sequencing (sRNA-seq). Low-quality reads and adaptors were removed to obtain clean reads. We used the miRDeep2 module to collect unique reads and then identify known miRNAs and predict novel miRNAs. So far, 196, 169 and 197 miRNAs had been identified in pre-J2s, adult males and females, respectively. PCA analysis of the sRNA-seq data clustered each developmental stage accordingly. We conducted differential expression analysis using edgeR to categorize miRNAs that are specifically or differentially expressed in female or male. We further predicted the potential target genes of differential expressed miRNAs using the miRanda program and the Meloidogyne incognita genome annotations. However, in the currently available Meloidogyne incognita genome annotations, 3'-UTR regions are available for only less than 40% of annotated genes in the genome which caused issues for miRNA target prediction. We are currently using the mRNA-seq data generated from the Objective 1 of this project and other publicly available M. incognita RNA-seq data to try to map and determine the 3'-UTR of more annotated genes in the genome. The information will be useful for predicting miRNA targets, GO enrichment analysis and selection of candidate miRNA genes for functional studies.
Publications
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