Source: UNIVERSITY OF CALIFORNIA, DAVIS submitted to NRP
CHARACTERIZATION OF ROOT-KNOT NEMATODES THAT INFECT RESISTANT TOMATO
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
NRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0186063
Grant No.
2001-35302-10135
Cumulative Award Amt.
(N/A)
Proposal No.
2000-02993
Multistate No.
(N/A)
Project Start Date
Dec 1, 2000
Project End Date
Nov 30, 2004
Grant Year
2001
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671
Performing Department
NEMATOLOGY
Non Technical Summary
Root-knot nematodes cause severe damage to many important crops. Although this nematode can infect a broad range of crops, there are considerable differences in the ability of different nematode isolates to reproduce on particular crop cultivars. The goal of this proposal is to identify genetic factors in the nematode that account for this variability. Specifically, a strain of root knot nematodes, VW5, that reproduces on tomato with the nematode resistance gene Mi will be compared to the strain VW4, which can not reproduce on tomato with Mi. Genomic DNA from these two nematode strains will be compared using powerful new selective techniques that allow identification of differences between nearly identical organisms. Once genetic differences are identified, the corresponding genes will be examined and tested to determine whether they are responsible for the ability of strain VW5 to reproduce on tomato with the Mi gene. How these genetic changes allow the nematode to reproduce on resistant tomato will be examined as will the mechanism by which the nematode has acquired the new ability. The proposed research should lead to a better understanding of how root-knot nematodes change their genomes to infect new host plants and should provide insight into strategies for more effective use of natural plant resistance genes for nematode control.
Animal Health Component
40%
Research Effort Categories
Basic
60%
Applied
40%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21231301040100%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
3130 - Nematodes;

Field Of Science
1040 - Molecular biology;
Goals / Objectives
Root knot nematodes have a very broad host range and infect a large number of crop plants, yet there is considerable variability within species in virulence and host range. The long range goal of this research is to understand how root-knot nematodes change genetically to acquire virulence against new hosts or hosts with specific resistance genes. The interactions of root-knot nematodes with tomato plants carrying the well-characterized nematode resistance gene, Mi, provides an excellent model system for these studies. The current objectives are to: 1)Identify molecular differences between a closely related pair of root-knot nematode strains that differ in their ability to reproduce on tomato with Mi and 2)Identify a gene that is responsible for the virulence difference between nematode strains VW4 and VW5.
Project Methods
The virulence of the nematode strain VW5 on tomato with the resistance gene Mi developed suddenly suggesting it was due to a single genetic event. This genetic event could be a mutation in a single gene, a deletion, an amplification or a genome rearrangement. Genomic differences between VW4 and VW5 will be sought using the technique cDNA AFLP. Candidate clones will be tested to see if they produce polymorphisms or intensity differences on DNA and RNA blots of paired nematode strains. Full length cDNAs corresponding to such polymorphisms will be obtained by 5' RACE. DNA and RNA blot analysis will be carried out to compare expression and genomic structure of this gene in nematode strains that differ in virulence. For tomato plants with Mi, the defense response is characterized by a localized hypersensitive response (HR). Candidate genes will be tested for their ability to elicit the HR in tomato with Mi by introducing these genes into Agrobacterium-based vectors that will express the candidate genes in the cytoplasm of tomato cells. Leaves of resistant tomato will be infiltrated to obtain transient expression of the introduced gene. An alternative approach is to express the candidate avirulence gene in a virus vector. The virus vector expressing the Mi-avirulence gene candidates will be used to infect tomato with and without Mi . The ability to infect tomato without, but not with, Mi should indicate identification of a nematode avirulence factor. If analysis of candidate differences suggest that the virulent strain has acquired new properties rather than lost a virulence factor, the corresponding genes will be cloned and the effects of their expression in plants will be examined. In either case, once the genes from nematodes responsible for differences in host range are identified, additional characterization will be initiated including determination of the localization and expression pattern of these genes in nematodes.

Progress 12/01/00 to 11/30/04

Outputs
The tomato gene Mi-1 confers resistance against several species of root-knot nematode. We have identified a strain of the root-knot nematode Meloidogyne javanica, VW5, which can reproduce on tomato carrying the Mi-1 gene. VW5 was isolated from a culture of the strain VW4, which cannot reproduce on resistant tomato. By screening for molecular polymorphisms, we identified a gene, CG-1, that is expressed in nematode juveniles of strain VW4 but is lacking in strain VW5. Our analyses indicated that the DNA corresponding to CG-1 missing in the virulent nematode VW5, suggesting that VW5 may have become virulent due to a deletion in the genome of VW4. Investigation of CG-1 in other Mi-virulent nematode strains indicated that CG-1 is also missing in some, but not all, of these strains. CG-1 does not encode a gene product with high similarity to others available in public databases. We attempted to determine whether CG-1 has a role in Mi-mediated resistance by using RNA silencing. Soaking avirulent nematodes in double-stranded RNA corresponding to CG-1 did result in gain of ability of these nematodes to reproduce on tomato carrying Mi-1. By propagating these nematodes on tomato with Mi-1, we have been able to maintain this phenotype for several generations. These results strongly suggest that CG-1 corresponds to the nematode avirulence gene, that is, it encodes a product that mediates recognition by the host plant with the Mi-1 gene. This is the first such gene identified in root-knot nematodes and an important resource for understanding how nematodes acquire virulence and for developing molecular tools to monitor this process. As another approach to understanding how pathogens such as nematodes can circumvent resistance, we investigated the effects of selected compounds on resistance mediated by Mi-1. We found that salicylic acid and ethylene synthesis are required for host resistance to nematodes mediated by Mi-1.

Impacts
Root knot nematodes cause major damage to thousands of crop species. Host resistance is a preferred control mechanism. However, the ability of nematodes to overcome resistance is an important constraint. It is necessary to understand how this asexual species can gain the ability to bypass host resistance in order to prolong the utility of resistance. The nematode avirulence gene that we have identified should provide us with a tool to monitor development of virulence of nematodes against the widely deployed resistance gene Mi-1and possibly to develop a diagnostic assay.

Publications

  • No publications reported this period


Progress 01/01/03 to 12/31/03

Outputs
The tomato gene Mi-1 confers resistance against several species of root-knot nematode. We had previously identified a nematode strain, VW5, which can reproduce on tomato carrying the Mi-1 gene. The strain VW5 was derived from a culture of the strain VW4, which can not reproduce on resistant tomato. We identified a gene, CG-1, that was expressed in nematode juveniles of strain VW4 but was lacking in strain VW5. Our analyses indicated that the DNA corresponding to CG-1 missing in the virulent nematode VW5, suggesting that VW5 may have developed as a virulent strain due to a deletion in the genome of VW4. Investigation of CG-1 in other Mi-virulent nematode strains indicated that CG-1 was also missing in some, but not all, of these strains. CG-1 does not appear to encode a gene product with high similarity to others available in public databases. We are attempting to determine whether CG-1 has a role in Mi-mediated resistance by using RNA silencing. As another approach to understanding how pathogens such as nematodes can circumvent resistance, we have investigated the effects of selected compounds on resistance mediated by Mi-1. We have found that salicylic acid is required for host resistance to nematodes mediated by Mi-1.

Impacts
Root knot nematodes cause major damage to thousands of crop species. Host resistance is a preferred control mechanism. However, the ability of nematodes to overcome resistance is an important problem. It is necessary to understand how this asexual species can gain the ability to bypass host resistance in order to prolong the utility of resistance.

Publications

  • Williamson, V.M. and C.A. Gleason. 2003. Plant-nematode interactions. Current Opinion in Plant Biology. 6:327-333.
  • Gleason, C.A. 2003. Comparison of two strains of Meloidogyne javanica differing in virulence on tomato with the resistance gene Mi, and identification of a polymorphism that correlates with avirulence. Ph.D. Thesis. University of California, Davis.
  • Branch, C., Hwang, C.F., Navarre, D.A. and Williamson, V.M. 2004. Salicylic acid is part of the Mi-1-mediated defense response to root-knot nematode in tomato. Mol. Plant Microbe Int. In press.


Progress 01/01/02 to 12/31/02

Outputs
The tomato gene Mi-1 confers resistance against several species of root-knot nematode. We had previously identified two closely related nematode strains, VW4 and VW5, that differ in their ability to reproduce on tomato with Mi; VW4 cannot reproduce on resistant tomato whereas VW5 can reproduce. We used the DNA marker technique, cDNA-AFLP, to identify differences in the gene expression pattern between these two strains. One clone, CG1, was found to be expressed in nematode juveniles of the avirulent strain VW4 but was lacking in the virulent strain VW5. DNA blot analysis revealed that this gene was a member of a gene family, one member of which was missing in strain VW5. The expression difference was confirmed by RT-PCR. We obtained the DNA sequence of this clone and have obtained additional clones of this region on the nematode genome. So far no strong similarity to genes of know function has been identified. More than one transcript size appears to be produced from this region of the genome and no long open reading frame has been identified. As an approach to understanding how pathogens such as nematodes may circumvent resistance, we have investigated the effects of selected compounds on resistance mediated by Mi-1. We have found that the plant hormone cytokinin can cause loss of resistance and that salicylic acid appears to be required for host resistance to function.

Impacts
Root knot nematodes cause major damage to thousands of crop species. Host resistance is a preferred control mechanism. However, the ability of nematodes to overcome resistance is an important problem. It is necessary to understand how this asexual species can change genetically to bypass host resistance in order to prolong the utility of this resistance.

Publications

  • No publications reported this period


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

Outputs
The gene Mi confers resistance against several species of root-knot nematode in tomato. We had previously identified two closely related nematode strains, VW4 and VW5, that differ in virulence on the resistance gene Mi. We used the PCR-based technique, cDNA-AFLP, to identify nematode genes that differ in expression pattern between these two strains. One clone, CG1, was expressed in juveniles of the avirulent strain VW4 but was lacking in the virulent strain VW5. DNA blot analysis revealed that this gene was a member of a gene family, one member of which was missing in strain VW5. The expression difference was confirmed by RT-PCR using several sets of primers. We have obtained the sequence of this clone and have used a number of techniques to obtain additional sequence of the gene. So far no strong similarity to genes of know function has been observed. As another approach to understanding virulence and avirulence, we have investigated the effects of selected compounds on resistance mediated by Mi. We have found that cytokinin can cause loss of resistance and that salicylic acid appears to be required for host resistance to function.

Impacts
Root knot nematodes cause major damage to thousands of crop species. Host resistance is a preferred control mechanism. However, the ability of nematodes to change to overcome resistance is an important problem. It is necessary to understand how this asexual species can change genetically to infect resistant plants in order to prolong the utility of host resistance.

Publications

  • No publications reported this period