Progress 12/01/06 to 11/30/10
Outputs
OUTPUTS: This project targets the plant disease resistance and resistance gene community. Results from our study will benefit applied as well as basic research on disease resistance genes and breeding for resistance to root-knot nematodes. Using funds from this grant we provided training to 2 postdoctoral fellows, 2 graduate students and 6 undergraduate students. Results from this work were presented in invited seminars: in 2007, 1) UC Davis, 2) Spanish National Research Council in Madrid, 3) Rothamsted Research, England, as well as an invited talk at the Annual joint meeting of American Phytopathological Society and Society of Nematologists, San Diego, CA. In 2008, at the fifth International Congress of Nematology, Brisbane, Australia. In 2009, in the Department of Plant Breeding, Wageningen University, Wageningen, The Netherlands. PARTICIPANTS: - Dr. Isgouhi Kaloshian (PI) - Kishor Bhattarai (graduate student) - Laura Cortada (visiting graduate student) - Olivia Desmond (postdoctoral fellow) - Sophie Mantelin (postdoctoral fellow) - Lana Khoury (undergraduate student) - Nhat Nguyen (undergraduate student) - Tori Owens (undergraduate student) - Ann Phan (undergraduate student) - Adam Puchalski (undergraduate student) - Chen Ung (undergraduate student) TARGET AUDIENCES: Results from this benefited both the agricultural community by providing new sources of root-knot nematode resistance, and the scientific community interested in disease resistance genes. This project also provided training for graduate and undergraduate students and postdoctoral fellows. Information from this work was used in an undergraduate genetic course and a graduate course in molecular plant-microbe interactions. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The three main findings of this project are: (1) We mapped seven MI-1 resistance gene homologues to the short arm of chromosome 6 of SOLANUM ARCANUM accession LA2157, a wild relative of cultivated tomato with heat-stable root-knot nematode (RKN) resistance. Three LA2157 MI-1 homologues (RH1, RH2, and RH4) co-localized to the MI-9 heat-stable resistance locus. Using tobacco-rattle based virus induced gene silencing to silence MI-homologues, we attenuated the MI-9 mediated heat-stable nematode resistance and demonstrated that Mi-9 gene is a homologue of MI-1. (2) To clone the four Mi-1 homologues that co-segregated with Mi-9, we developed a genomic DNA library derived from LA2157 leaf tissue in Lambda FIX II phage vector. Using several distinct MI-1 probes and different rounds of screens, a total of 16 unique clones were identified. Twelve of the clones overlap to form a contig of 44.5 kb. This contig contains two full-length NBS-LRR encoding genes with high homology to MI-1.2, MI-1.1, as well as a pseudogene similar to MI-1.3, genes present in the MI-1 locus. In addition, four clones that did not overlap with this contig were identified. Of these, two contained partial MI-1 type sequences and are likely to represent a second cluster of MI genes from the short arm of chromosome 6 in LA2157. Sequence analysis of the LA2157 44.5 kb contig compared to known sequences from S. PERUVIANUM have revealed a high degree of homology within the MI coding sequences and low homology in non-coding sequences between genes. The organization of the LA2157 MI genes is similar to previously characterized MI genes and consists of 2 introns and 2 exons, with the first intron located upstream of the coding sequence and the second intron within the coding sequence. The first intron is 75 bp long and located 45 bp after the start codon is conserved in the LA2157 homologs. The coding sequence of the MI genes from LA2157 and S. PERUVIANUM is highly conserved. There is between 93-98% identity at the nucleotide level and between 87-97% identity and 92-98% similarity at the amino acid level. (3) LA2157 cDNA was used as a template for PCR-based amplification and cloning of Mi homologs. Clones were restriction digested for fingerprinting and categorized into groups. A subset was sequenced.18 clones were confirmed as MI homologs, and 14 were partially sequenced. One of the LA2157 clones with homology to MI-1.1 and a predicted heat-stable motif was cloned into the expression vector pMOA33 behind the 35S promoter, and transformed using AGROBACTERIUM TUMEFACIENS into a commercial tomato cultivar that is susceptible to root-knot nematode. Thirteen independent transgenic lines were developed and transgenic plants (T1), expressing the transgene, were evaluated for heat-stable nematode resistance. None of the transgenic plants conferred heat-stable resistance to RKN.
Publications
- (1) Jablonska, B., J. S. S. Ammiraju, K. Bhattarai, O. Martinez de Ilarduya, S. Mantelin, P. A. Roberts, and I. Kaloshian. 2007. The MI-9 gene from SOLANUM ARCANUM conferring heat-stable resistance to root-knot nematodes is a homologue of MI-1. Plant Physiol. 143: 1044-1054. (2) Cortada, L., F. J. Sorribas, C. Ornat, I. Kaloshian, and S. Verdejo-Lucas. 2008. Variability in infection and reproduction of MELOIDOGYNE JAVANICA on tomato rootstocks with the MI resistance gene. Plant Pathology 57:1125-1135.
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Progress 12/01/08 to 11/30/09
Outputs
OUTPUTS: Library screening: The MI-9 gene conferring heat-stable resistance to root-knot nematodes (MELOIDOGYNE spp.) has been identified in SOLANUM ARCANUM accession LA2157, a wild relative of cultivated tomato (SOLANUM LYCOPERSICUM). Without knowing the exact sequence of this gene, previous work has shown that it is a homolog of the MI-1.2 gene that confers heat-sensitive resistance to root-knot nematodes and encodes a coiled-coil (CC) nucleotide binding site (NBS) and leucine-rich repeat type protein. Four MI-1 homologs cosegregate with the heat-stable resistance all located on the short arm of chromosome 6 in the same genetic interval as MI-1.2. In this reporting period, we have continued screening of the LA2157 gDNA Lambda library using the CC, NBS, or intron-1 as probes. Most positive clones have been sequenced. Sequence analysis was performed using BLAST and Vector NTI to compare sequences between S. ARCANUM and SOLANUM PERUVIANUM, the source of the heat-sensitive resistance. Expressing a MI homolog in tomato: One of the LA2157 clones with homology to MI-1.1 and a predicted heat-stable motif has been cloned into the expression vector pMOA33, and is currently being transformed using AGROBACTERIUM TUMEFACIENS into a commercial tomato cultivar that is susceptible to root-knot nematode infection. Cloning MI homologs from cDNA: LA2157 gDNA or cDNA has been used as a template for PCR-based cloning of Mi homologs into the TOPO vector. Clones were restriction digested for fingerprinting and to categorize them into groups. A subset was then sequenced. PARTICIPANTS: This project provided training to a graduate student and a postdoctoral fellow. It also provided funds for the PI to attend the International Congress for Molecular Plant-Microbe Interactions in Quebec, Canada. TARGET AUDIENCES: Several disease resistance genes in diverse crops to various pathogens are heat-sensitive. Therefore, diverse groups working in the plant industry are interested in this research such as: farmers, seed companies and academia. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
A total of 3 screens have been completed resulting in the identification of 16 unique clones with sequences from the short arm of chromosome 6 where the MI homologs are situated. Twelve of the clones overlap to form a contig of 44.5 kb. This contig contains two full-length NBS-LRR encoding genes with high homology to MI-1.2, MI-1.1, as well as a pseudogene similar to MI-1.3. In addition, four clones that don't overlap with this contig have been identified. Of these, two contain partial MI-1 type sequences and are likely to represent a second cluster of MI genes from the short arm of chromosome 6 in LA2157, similar to that observed in S. PERUVIANUM and S. LYCOPERSICUM. Sequence analysis: Sequence analysis of the LA2157 44.5 kb contig compared to known sequences from S. PERUVIANUM have revealed a high degree of homology within the MI coding sequences and low homology in non-coding sequences between genes. The organization of the LA2157 MI genes is similar to previously characterized MI genes and consists of 2 introns and 2 exons, with the first intron located upstream of the coding sequence and the second intron within the coding sequence. The first intron has previously been used to distinguish between the homologs due to variations in length, and this feature is also conserved in the LA2157 homologs. Intron 2 is highly conserved between all of the MI homologs and this is maintained for the LA2157 homologs. It is 75 bp long and located 45 bp after the start codon. The position and sequence of the TATA-box (TATTTATA) and the start codon is identical between LA2157 and S. PERUVIANUM. The coding sequence of the MI genes from LA2157 and S. PERUVIANUM is highly conserved. There is between 93-98% identity at the nucleotide level and between 87-97% identity and 92-98% similarity at the amino acid level. The region upstream of the transcript start site is conserved between the two LA2157 clones, and also conserved with S. PERUVIANUM MI genes. Interestingly, there seems to be a roughly 1kb insertion directly upstream of the transcript start site of one of the LA2157 that has little homology to the other homolog or orthologs. The untranscribed region of homology in this clone is further upstream of this insertion. The degree of homology from -1 to -300 of the transcript start site is between 87-95%, while homology drops suddenly in the next 1kb from -301 to -1300 to 34-45%. PCR based cloning: A total of 24 clones were screened, 18 clones have been confirmed as MI homologs, and 14 of these have been partially sequenced. As expected most homologs were isolated several times including the two identified in the genomic lambda library screens. In addition, three full-length unique clones, not found in the library screen, have been identified and fully sequenced. Dissemination: Several disease resistance genes in diverse crops to various pathogens are heat-sensitive. Identifying the nature of heat stability of MI-9 will allow the development of crops with heat-stable resistance which is critical in the current global warming trend. Results from this work were present at invited seminars in the US, Belgium and the Netherlands.
Publications
- Cortada, L., F. J. Sorribas, C. Ornat, I. Kaloshian, and S. Verdejo-Lucas. 2008. Variability in infection and reproduction of MELOIDOGYNE JAVANICA on tomato rootstocks with the MI resistance gene. Plant Pathology 57:1125-1135
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Progress 12/01/07 to 11/30/08
Outputs
The MI-9 gene conferring heat-stable resistance to root-knot nematodes (MELOIDOGYNE spp.) has been identified in SOLANUM ARCANUM accession LA2157, a wild relative of cultivated tomato. Without knowing the exact sequence of this gene, previous work has shown that it is a homolog of the MI-1 gene that confers resistance to root-knot nematodes. Four MI-1 homologues cosegregate with the heat-stable resistance all located on the short arm of chromosome 6 in the same genetic interval as MI-1. In this reporting period, we have worked to isolate and sequence these MI-1 homologues by constructing and screening a genomic DNA library derived from LA2157 leaf tissue. This genomic library was developed using the Lambda FIX II phage vector. LA2157 DNA was purified, partially restricted with SAU3A, ends were partially filled in, ligated into Lambda FIX II vector, packagedOur work has produced a genomic DNA library from the wild tomato species SOLANUM ARCANUM accession LA2157, that is known to have heat-stable resistance to root-knot nematodes. Screening of this library has identified 11 unique clones containing sequences with homology to the nematode resistance gene MI. and propagated on XL1-Blue MRA (P2) E. COLI cells. The primary library was tittered and found to contain 1.91x106 plaque forming units per mL, well within the range required for screening. The insert size ranged between 10-23 Kb with an average insert size around 14 Kb. Primary, secondary and tertiary screens of this phage library was performed using a probe (IK3-3), from the nucleotide-binding site of MI-1, previously used to identify MI homologs in several tomato varieties, including LA2157 (Jablonska, et al. 2007). A total of 11 unique clones with positive IK3-3 probe hybridization have been identified. To further characterize these phage clones, the inserts were isolated and subcloned into pBS-SK (-) vector. Subsequently, the ends of these clones were sequenced and aligned to the short arm of chromosome 6. Preliminary sequencing results suggest that the genomic region from the first intron of MI to the centromere of chromosome 6 has been captured in these library screens. To expand the characterized region to include regulatory sequences located upstream of intron 1, the library is currently being re-screened with a mixture of probes representing the different intron 1 of the four MI-1 homologues segregating with the heat-stable resistance in LA2157. Participants: Not relevant to this project. Target Audiences: The availability of a LA2157 genomic library will benefit the scientific community as this wild tomato relative contains additional disease resistance genes. This project also provided training for a graduate student, a postdoctoral fellow as well as an undergraduate student. Project Modifications: Not relevant to this project.
Impacts
Our work has produced a genomic DNA library from the wild tomato species SOLANUM ARCANUM accession LA2157, that is known to have heat-stable resistance to root-knot nematodes. Screening of this library has identified 11 unique clones containing sequences with homology to the nematode resistance gene MI.
Publications
- No publications reported.
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Progress 12/01/06 to 11/30/07
Outputs
OUTPUTS: Root-knot nematodes (MELOIDOGYNE spp.) are root endoparasites of numerous crops worldwide and are the most damaging nematode pest in agriculture. Resistance conferred by the MI-1 gene from SOLANUM PERUVIANUM is effective and widely used for limiting root-knot nematode yield loss in tomato (SOLANUM LYCOPERSICUM), but the resistance is ineffective at soil temperatures above 28C. Previously, heat-stable resistance to root-knot nematodes was identified in SOLANUM ARCANUM accession LA2157, a wild relative of tomato. The heat-stable resistance was conferred by a single dominant gene MI-9 and was mapped to the short arm of chromosome 6, in a genetic interval as MI-1 and the CLADOSPORIUM FULVUM resistance gene CF2. We developed a fine map of the MI-9 region by nematode screens and marker analysis of an F2 population and derived F3 families from resistant LA2157 x susceptible LA392. We designed primers flanking an intron in MI-1 that were expected to amplify other MI-1 homologues
because of high degree of sequence conservation. These primers were then utilized to determine a fingerprint of MI-1 homologues, based on intron-length polymorphism. We had performed preliminary experiments to determine whether MI-9 is a homologue of MI-1. We used virus-induced gene silencing and tobacco rattle virus vector (TRV) and targeted transcripts of MI-1 homologues for degradation in LA2157. Results from this work were presented in seminars at: 1) UC Davis, 2) Spanish National Research Council in Madrid, 3) Rothamsted Research, England, 4) American Phytopathological Society and Society of Nematologists Annual joint meeting.
PARTICIPANTS: Sophie Mantelin - postdoctoral fellow; Kishor Bhattarai - graduate student
TARGET AUDIENCES: Results from this will benefit both the agricultural community by providing new sources of root-knot nematode resistance, and the scientific community by identifying the nature of the heat-stable resistance. This project also provides training for a graduate student and a postdoctoral fellow. Information from this work is used in an undergraduate genetic course and a graduate course in molecular plant-microbe interactions.
PROJECT MODIFICATIONS: none
Impacts
Our work identified seven MI-1 homologues in the mapping parents and all seven homologues mapped to the short arm of chromosome 6. Three MI-1 homologues from LA2157 and one from LA392 co-localized to the MI-9 region. In addition, our results suggested that TRV-MI construct could silence MI-9-mediated heat-stable resistance. In most LA2157 plants infiltrated with the TRV-MI construct, MI-9 meditated heat-stable root-knot resistance was compromised at 32C indicating that the heat-stable resistance is mediated by a homologue of MI-1 In the past decade, a large number of plant disease resistance genes were cloned and most encoded proteins with similar structural motifs. Substantial effort and resources were dedicated for cloning and identification of these resistance genes. Our work indicates that using recent development in technology, such as virus-induced gene silencing, it is possible to identify the nature of a resistance gene before cloning and in a short time
period.
Publications
- Jablonska, B., J. S. S. Ammiraju, K. Bhattarai, O. Martinez de Ilarduya, S. Mantelin, P. A. Roberts, and I. Kaloshian. 2007. The MI-9 gene from SOLANUM ARCANUM conferring heat-stable resistance to root-knot nematodes is a homologue of MI-1. Plant Physiol. 143: 1044-1054.
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