VARIABILITY, ADAPTATION AND MANAGEMENT OF NEMATODES IMPACTING CROP PRODUCTION AND TRADE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: NEW
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1018832
• Project No.: CA-R-NEM-5158-RR
• Multistate No.: W-4186
• Project Start Date: Feb 20, 2019
• Project End Date: Sep 30, 2023

Project Director
Kaloshian, IS.

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

Performing Department
Nematology, Riverside

Non Technical Summary
Root-knot nematodes are the most important agricultural nematodes in the US and worldwide. With an extensive and variable host range, it is difficult to predict what hosts they will parasitize. In addition, current resistances sources in crops are losing effectiveness and to screen plants with nematodes is a labor-intensive process requiring large space in greenhouses.We will optimize a laboratory-based infection system to accelerate the screen of roots with these nematodes. A CRISPR-Cas9 modified tomato roots, to create enhanced nematode resistance, will be used for these studies. We will also develop and implement the use of DNA-based nematode infection evaluation to overcome difficulties with quantification of nematode infection rates.

Animal Health Component

0%

Research Effort Categories

Basic

90%

Applied

10%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	1460	1120	100%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1460 - Tomato;

Field Of Science
1120 - Nematology;

Keywords

tomato

crisp-cas9

enhanced resistance

root-knot nematodes

infection rate

Goals / Objectives
1. Characterize genetic and biological variation in nematodes relevant to crop production and trade.

Project Methods
The goal of this work is to study enhanced resistance phenotype in tomato and identify its breadth against root-knot nematode (RKN) Meloidogyne incognita populations and additional RKN species. The following aims and approaches will be used to accomplish this goal.Aim 1. Establish a hairy root system to use CRISPR -Cas9 system to modulate resistance against root-knot nematodes. Since tomato transformation takes a long time to develop, we will develop hairy root transformation system to evaluate CRISPR-Cas9 vectors to mutate a negative regulator of plant immunity. To accomplish this, we will develop hairy root transformation system, using Rhizobium rhizogenes (Agrobacterium rhizogenes), using tomato varieties desirable by farmers. Although hairy root of tomato is well established, such infections are not always highly successful with different tomato cultivars. We will use a couple of commercially desirable tomato cultivars and a few R. rhizogenes strains, known to infect tomato, to choose the best combination. Once an optimum hairy root transformation is discovered, the R. rhizogenes strain will be transformed with CRISPR-Cas9 vector(s) to mutate a negative regulator of immunity. Effective mutation will be evaluated using both molecular marker as well as gene expression analyses and roots with full mutated negative regulator gene will be selected for further studies.Aim 2. Evaluate the breadth of the resistance to nematodes. Tomato hairy roots, mutated for the negative regulator of immunity, will be evaluated with different populations of Meloidogyne incognita as well as additional species of RKN, such as, Meloidogyne javanica, Meloidogyne arenaria and Meloidogyne hapla. Additional nematode species that could infect tomato will also be considered for infection evaluation. Nematodes will be maintained on tomato plants in greenhouse conditions. Nematode eggs will be extracted, floated on sugar gradient and surface sterilized using beach. Surface sterilized eggs will be hatched under sterile conditions and infected-stage juveniles used to infect the R. rhizogenes transformed roots.Aim 3. Develop a quantitative approach to evaluate nematode infections of hairy roots in cultured plates. Hairy root, as the name indicates, develop a tangle of roots with numerous branches. To quantitatively evaluate nematode infection, we will use a quantitative-PCR (qPCR) approach to quantifying both tomato and nematode DNAs using their respective housekeeping genes. Hairy roots, grown in plates, infected with equal number of infective-stage juvenile nematodes will be used for DNA extraction. Using species-specific primers in PCR, the quantity of DNAs from the respective organisms, collected from a single plate, will be determined and accurate quantification of nematode infection rates will be calculated. All experiments will be performed with biological replicates and repeated in time to provide statistical relevant data.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:Our effort to disseminate our results was hindered because of the pandemic restrictions. Results were presented at the annual meeting of the multistate group participants only. Changes/Problems:The COVID19 pandemic restrictions in Riverside county (CA) has affected our progress and effective management of this project and its goals. What opportunities for training and professional development has the project provided?The project has provided training for a postdoctoral fellow. How have the results been disseminated to communities of interest?Our effort to disseminate our results was hindered because of the pandemic restrictions. Results were presented at the annual meeting of the multistate group participants only. What do you plan to do during the next reporting period to accomplish the goals?Homozygous CRISPR plants will be identified and used for evaluation with root-knot nematode. Additional CRISPR-edited tomatoes are being developed as well hoping for homozygous edited plants.

Impacts
What was accomplished under these goals? Biotechnology offers new approaches to nematode control and will reduce the reliance on nematicides, which are often expensive. We have identified a negative regulator of root-knot nematode immunity in Arabidopsis and have shown that the absence of this gene results in over 50% decrease in RKN infection. To assess the role of this gene in tomato, they developed CRISPR-Cas9 constructs to target the tomato putative ortholog(s) of the Arabidopsis gene. Several primary tomato transformants were obtained and a few made it to maturity. A number of putative transgenic plants were lost due to lack of daily care because of the pandemic lockdown. Sequence analysis indicated that the CRISPR edited plants had point mutations and were all heterozygous for the mutations. All transgenic plants that made it to maturity were able to set fruits with seeds. The seeds will be planted to obtain homozygous mutants for evaluation with RKN.

Publications

Progress 02/20/19 to 09/30/19

Outputs
Target Audience:This project targeted the nematology research communities. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project provided training to one undergraduate student and partial funding for a postdoctoral fellow. Professional development for a postdoc included presentation of a poster at the Annual meeting of American Society of Plant Biologists, San Jose, CA, in August 2019. How have the results been disseminated to communities of interest?Results from this project were presented at a number of scientific meetings related to nematology and plant immune biology. These included oral or poster presentations: at the XVIII Congress of the International Society for Molecular Plant-Microbe Interactions, July 2019; Annual meeting of the Society of Nematologists, July 2019; and at the Annual meeting of the American Society of Plant Biologists, August 2019. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Plants recognize nematode infections during root penetration, before initiating an elaborate feeding site characteristic of sedentary endoparasitic nematodes, and initiating a strong immune response. This immune response, known as pattern-triggered immunity, is tightly regulated. Tight regulation is necessary to control runaway immunity that may cause uncontrolled cell death or autoimmune disease. Such controls, also known as negative regulators of immunity, have been identified in Arabidopsis thaliana, orthologs are which are also present in crops including tomato. The absence of one such negative regulators results in enhanced resistance to the root-knot nematode, Meloidogyne incognita, in Arabidopsis. To develop a similar resistance in tomato, tomato orthologs of the Arabidopsis gene were targeted for mutations using CRISPR-Cas9 gene editing. CRISPR-Cas9 vectors used for editing tomato were used to develop constructs to edit two different tomato homologs of the negative regulator of immunity. Constructs targeting the individual genes or both genes together, using two guide RNAs for each, were developed and used in Agrobacterium/Rhyzobium rhizogenes transformation of tomato cotyledons. Hairy roots induced by A. rhizogenes transformation were evaluated for deletions in the targeted genes using PCR. Our results indicated high efficiency in deletion in one of the targeted genes and lower efficiency in the second gene. No transgenic roots were identified with deletions in both genes suggesting either low mutation efficiency or lethal phenotype when both genes are eliminated. The successful constructs are currently being used to develop stable transgenic gene edited tomato plants for evaluation with nematodes.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Kaloshian, I and M. Teixeira. 2019. Advances in plant?nematode interactions with emphasis on the notorious nematode genus Meloidogyne. Phytopathology 109:1988-1996.19:239.