Progress 10/01/07 to 09/30/12
Outputs
OUTPUTS: This project targets the entomology, nematology, and plant immune biology research community. Results from our study were presented at a number of scientific meetings related to entomology, nematology, and plant immune biology. These include: a total of 12 presentations at International Meetings; five of which were invited talks (International Nematology Congress, two talks, Brisbane, Australia 2008; European Society of Nematology meeting, Vienna, Austria, 2010; Hempiteran-Plant Interactions Symposium, Piracicaba, Brazil, 2011; International Plant and Animal Genome XX, San Diego, USA, 2012). Four presentations at National meetings two of which were invited talks (Entomological Society of America, San Diego, 2010; American Society of Plant Biologists, Austin, 2012). In addition to three seminars and one presentation in the West Coast Undergraduate Research Conference, San Diego 2008. PARTICIPANTS: Isgouhi Kaloshian- principle investigator; Graeme Kettles- Postdoctoral fellow; Barbara Jablonska- Staff research Associate; Hagop Atamian- graduate student; Hsuan-Chieh Peng- graduate student; Ritu Chaudhary- graduate student; Anthony Beavers- undergraduate student; Erika Galvez- undergraduate student. TARGET AUDIENCES: This project targets the nematology, plant pathology, entomology and plant immune biology research community. Results from our study will benefit research on applied as well as basic entomology/nematology/plant immune biology. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
1) Resistance to potato aphid (MACROSIPHUM EUPHORBIAE) in tomato (SOLANUM LYCOPERSICUM) is conferred by the resistance (R) gene MI-1. Early in both compatible and incompatible interactions, potato aphid feeding induces expression of ethylene (ET) biosynthetic genes and ET production. We used genetic and pharmacologic approaches to investigate the role of ET in basal defense and MI-1-mediated resistance to potato aphid in tomato. Bioassays were performed using plants impaired in ET biosynthesis or perception using virus-induced gene silencing (VIGS), the Never ripe mutant, and 1-methylcyclopropene treatment to block ET receptors. Interestingly, impairing ET signaling or biosynthesis did not compromise MI-1-mediated resistance but it did decrease susceptibility to aphids in compatible host. Our results indicate that ET may not play an important role in MI-1 resistance to aphids but modulates host basal defense enhancing its susceptibility to the aphid. 2) Using gene expression analysis, we identified WRKY transcription factor genes related to Arabidopsis AtWRKY72, named SlWRKY72a and SlWRKY72b, differentially regulated during MI-1-mediated incompatible interactions with root-knot nematode (RKN) and potato aphid. Using VIGS to suppress each gene individually or co-silenced both in tomato, we demonstrated that both genes are required for Mi-1 resistance and basal defense to RKN and potato aphids. Both WRKY genes were dispensable for bacterial pathogen PSEUDOMONAS SYRINGAE resistance mediated by the R gene PTO. Functional characterization of Atwrky72 mutants showed that this gene is also important for basal defense to RKN and oomycetes HYALOPERONOSPORA ARABIDOPSIDIS (HPA) Noco but not to the aphid MYZUS PERSICAE. WRKY72 was also dispensable for RPS2, RPM1 and RPP4 R gene-mediated defenses in Arabidopsis. Microarray analysis of Col-0 and wrky72 mutants indicated that HPANoco infection triggered massive transcriptional reprogramming in Col-0. About 17% of these genes were regulated in an AtWRKY72 dependent manner. The majority of these genes were not SA responsive indicating that AtWRKY72 controls aspects of basal defense independent of SA-dependent immune processes. 3) The plant receptor-like kinase SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK)3 is required for microbe-associated molecular pattern triggered immunity. Using TRV-VIGS a member of this family from tomato SlSERK1 was identified to be required for full function of Mi-1. In this process, we cloned and named all three SlSERK members: SlSERK1, SlSERK3A and SlSERK3B. Suppressing SlSERK1 transcript levels in resistant plants revealed a role for SlSERK1 in MI-1 resistance to potato aphids but not to RKN. In addition, Mi-1-dependent SlWRKY72 gene regulation was compromised in SlSERK1 silenced plants placing SlSERK1 in MI-1 signaling pathway. Silencing SlSERK1 in susceptible tomato did not reduce susceptibility to aphids indicating that SlSERK1 is unlikely to be a virulence target. SlSERK1 is an active kinase, mainly localized at the plasma membrane. This work identified a critical early component of MI-1 signaling and demonstrated a role for receptor kinases in NB-LRR mediated immunity.
Publications
- Atamian, H. S., T. Eulgem, and I. Kaloshian. 2012. SlWRKY70 is required for Mi-1-mediated resistance to aphids and nematodes in tomato. Planta 235:299-309.
- Atamian, H., P. A. Roberts, and I. Kaloshian. 2012. High and low throughput screens with root-knot nematodes Meloidogyne spp. J. Vis. Exp. 61, e3629.
- Cortada, L., S. Mantelin, S. Verdejo-Lucas, I. Kaloshian. 2012. Marker analysis for detection of the Mi-1.2 resistance gene in tomato hybrid rootstocks and cultivars. Nematology 14:631-642.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: This project targets the entomology, nematology, and plant immune biology research community. Results from our study will benefit research on applied as well as basic plant entomology/nematology/plant immune biology. Using funds from this grant we provided in 2011 training to the following individuals: - Hagop Atamian (graduate student) - Hsuan-Chieh Peng (graduate student) - Anthony Karapetians (undergraduate student) - Chen Ung (undergraduate student) - Esther Cai (undergraduate student) - Joseph Avila (undergraduate student) - Tatev Kirakosyan (undergraduate student) Results from this study have been presented in the following venues: -"Dissecting Mi-1-mediated resistance to aphids and nematodes", July 2011, Hempiteran-Plant Interactions Symposium, Piracicaba, Brazil. Invited talk. - "A systematic nomenclature for WRKY transcription factors", August 2011, America Society of Plant Biologists Annual Meeting. Minneapolis, MN. - "Mi-1-mediated potato aphid and root-knot nematode resistance: The role of pattern recognition coreceptors and more", Department of entomology, Purdue University, West Lafayette, IN. Invited seminar. PARTICIPANTS: Isgouhi Kaloshian- principle investigator Hagop Atamian- graduate student Hsuan-Chieh Peng- graduate student Sophie Mantelin- former postdoctoral fellow Beibei Lee- former postdoctoral fellow TARGET AUDIENCES: This project targets the plant pathology, entomology and plant immune biology research community. Results from our study will benefit research on applied as well as basic entomology/nematology/plant immune biology. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Phylogenetic analysis of the putative protein sequences of SlSERK3A and B indicated that both are homologues of the Arabidopsis SERK3/BAK1 (89% sequence identity). SlSERK3A and B are 10,874bp and 7,963bp in size, respectively. They are located on chromosome 10 and 1, respectively. The predicted SlSERK3A (616 aa, 68.28 kDa) and SlSERK3B (618 aa, 68.27 kDa) proteins have domains characteristic of SERK proteins including a signal peptide, a leucine zipper, 5 leucine-rich repeats domain, a Ser/Pro-rich region, two SPP motives, a single membrane-spanning domain and 11 conserved subdomains of a putative Ser/Thr protein kinase, followed by a short C-terminal tail. Additional features indicated that these proteins are transmembrane protein likely anchored to the plasma membrane (PM). The subcellular localization of SlSERK3A and SERK3B determined using confocal microscopy, verified this localization. The fluorescent signal of the SlSERK3A::GFP and SlSERK3B::GFP appeared to be localized mainly at the PM. The SlSERK3A and SlSERK3B transcripts were detected in all plant tissues tested albeit with variable levels of expression. The gene-specific VIGS silencing construct silenced the targeted genes. Co-silencing both SlSERK3A and B resulted in necrotic lesion in leaves suggesting roles for both genes in cell death. This TRV construct that co-silenced both genes was not used for bioassays. Using gene-specific TRV-VIGS constructs silencing an individual SERK3 gene, no role for either SERK3A or B was detected in MI-1-mediated resistance to potato aphids or RKN. Silencing SERK3A in susceptible Moneymaker plants resulted in enhanced susceptibility to RKN indicating a role in RKN basal defense. A total of 52.6 million paired-end reads were generated (total length 152 nucleotides) were generated, corresponding to 6.54 Gb sequence yield. The reads were assembled into 16,569 contigs (N50=2012 bp). About 10,000 have a length of more than 1,000 bp. About 66% of the assembled transcripts showed significant similarity (E value < 1e-3) to proteins in the UniProt database. The alignment of the assembled contigs to the predicted transcripts of the pea aphid (ACYRTHOSIPHUM PISUM), the genome of which has been sequenced, showed more than 70% coverage of 5318 pea aphid transcripts. Annotation of the sequences using GO terms revealed that the majority of the contigs were of nuclear origin followed by those localized to the plasma membrane. The highest category of molecular function was that of catalytic activity followed by protein binding, while the biological processes were represented by organismal development and nucleic acid metabolism at the highest frequencies.
Publications
- Bao, E., T. Jiang, I. Kaloshian, and T. Girke. 2011. SEED: efficient clustering of next-generation sequences. Bioinformatics 27:2502-2509.
- Kaloshian, I., O. Desmond, and H.S. Atamian. 2011. Disease resistance genes and defense responses during incompatible interactions. In: Genomics and Molecular Genetics of Plant-Nematode Interactions. J. Jones, G. Gheysen and C. Fenoll, eds. Springer, Dordrchet
- Mantelin, S., H.-C. Peng, B. Li, H. S. Atamian, F. L.W. Takken, and I. Kaloshian. 2011. The receptor-like kinase SlSERK1 is required for MI-1-mediated resistance to potato aphids in tomato. The Plant J. 67:459-471.
- Seifi, A., I. Kaloshian, J. Vossen, D. Che, K. K. Bhattarai, J. Fan, Z. Naher, A. Goverse, F. Tjallingii, P. Lindhout, R. Visser, and Y. Bai. 2011. Linked, if not the same, MI homologue(s) confer(s) resistance to tomato powdery mildew and root-knot nematodes. Mol. Plant-Microbe Interact. 24:441-450.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: In tomato, resistance (R) to root-knot nematodes (RKN) and potato aphids is conferred by the MI-1 gene. To identify additional components of the MI-1-mediated signaling pathway, we performed a high throughput suppressor screen of the MI-DS4-dependent hypersensitive response (HR) in NICOTIANA BENTHAMIANA. MI-DS4 is an autoactive form of MI-1. We used a tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) approach and a normalized cDNA library in AGROBACTERIUM TUMEFACIENS derived from mixed leaf tissues of tobacco mosaic virus (TMV)-resistant NN and TMV-susceptible N. BENTHAMIANA plants exposed to TMV. Four weeks after TRV inoculation of N. BENTHAMIANA, two leaves were spot infiltrated with A. TUMEFACIENS expressing MI-DS4 or autoactive PTO R-protein PTOL205D that confers resistance to PSEUDOMONAS SYRINGAE. A single plant per construct was used and HR symptoms recorded 5 days after spot-infiltration. Clones resulting in altered HR phenotype were re-assayed. One of the clones identified in the suppressor screen is SOMATIC EMBROYGENESIS RECPETOR KINASE 1 (SERK1). The role of SERK1 was evaluated in MI-1-mediated resistance in tomato. Gene-specific TRV-SERK1 VIGS construct was developed and tomato seedlings were treated with the TRV-SERK1 by agroinfiltration. For RKN assays, three weeks after TRV agroinfiltration, tomato plants were inoculated with 10,000 infective-stage juveniles per pot and maintained at 21-27C. Both Motelle (MI-1/MI-1) and Moneymaker (mi/mi) plants were used. Eight weeks after inoculation, nematode reproduction was evaluated by staining the roots in erioglaucine. Roots were sampled for gene expression analysis. For aphid assays, about five weeks after TRV agroinfiltration Motelle plants and controls were infested with 50 apterous adults and nymphs in a cage on a single leaflet and 4 leaflets per plant. About 14 days later, the number of aphids was counted. For basal defense assays, TRV agroinfiltrated Moneymaker plants were infested with age-synchronized one-day-old adult aphids. Three aphids were deposited on a single leaflet 4 weeks after agroinfiltration. Three leaflets per plants were infested and 8 plants per construct were used. Aphid survival and fecundity were monitored daily. Aphid assays were performed in a pesticide free greenhouse at 21-27C. At the end of experiments, assayed leaflets were collected for gene expression analysis. IN TRV-SERK1 treated tissue the SERK1 transcript level was evaluated by quantitative real-time PCR. The genomic and cDNA sequences of SERK1 were identified and the structure of the gene and the deduced protein were analyzed. In addition, the kinase domain was expressed in a heterologous system as a GST-fusion protein, GST-SlSERK1Kinase, for phosphorylation assays. Moreover, the subcellular localization of SlSERK1 was determined in vivo using a translational fusion to green fluorescent protein (GFP) a 35S-SERK1::GFP construct. Furthermore, the expression of SERK1 transcripts was evaluated in different tissue including mature leaf, young leaf, stems, roots, root tips, and flower. PARTICIPANTS: Isgouhi Kaloshian- principle investigator, Hagop Atamian- graduate student, Kishor Bhattarai- former graduate student, Sophie Mantelin- postdoctoral fellow. TARGET AUDIENCES: Results were presented at the Center for Disease Vector Research Symposium, UCR, March 2010, The European Society of Nematologists meeting in Vienna, Austria, September, 2010 and the Entomological Society of America Annual Meeting in San Diego, CA, December 2010. PROJECT MODIFICATIONS: none
Impacts
Over 2100 independent TRV clones were screened and 19 non-redundant cDNAs resulted in attenuation of the MI-DS4-mediated HR but not PTOL205D-mediated HR. One of these clones, P47-G3, revealed significant homology to proteins encoding SOMATIC EMBROYGENESIS RECPETOR KINASE 1 (SERK1). Silencing this protein did not attenuate the HR mediated by a number of R-proteins expressed with their cognate avirulence partners indicating that SERK1 is specifically required for MI-DS4 HR. Tomato has three members of the SERK1 gene family. Using SERK1 gene-specific TRV-VIGS construct we showed a role for SERK1 in MI-1-mediated resistance to potato aphids but not to RKN. In addition, silencing SERK1 in susceptible Moneymaker plants did not result in enhanced susceptibility to the aphid indicating no role in basal defense. The tomato SERK1 gene, SlSERK1, is located on chromosome 4 and is 6,297 bp in length. The protein coding sequence (CDS) is 1,890 bp. Comparing the CDS and the genomic sequences of SlSERK1 indicated the presence of 11 exons. The predicted SlSERK1 protein (629 amino acids, 69.33 kDa) has domains characteristic of SERK proteins including a signal peptide, with a putative cleavage site between amino acids 30 and 31, a leucine zipper followed by five successive leucine-rich repeatsdomains, the Ser/Pro-rich region, a distinctive feature of SERK family members containing two SPP motives, a single membrane-spanning domain and 11 conserved subdomains of a putative Ser/Thr protein kinase, followed by a short C-terminal tail. Consistent with high sequence identities, the deduced SlSERK1 closely clustered with known Solanaceae and Arabidopsis SERK proteins: SpSERK1 (99.5%), StSERK1 (98.7%), AtSERK1 (84.8%) and AtSERK2 (87.6%). Analysis of the SlSERK1 protein sequence and the hydrophobicity profile predicted a single TM helix between the receptor-like part and the kinase domain suggesting that SlSERK1 is a transmembrane protein likely anchored to the plasma membrane (PM) analogous to other SERK proteins. To verify this, the subcellular localization of SlSERK1 was determined using confocal microscopy. The fluorescent signal of the SlSERK1::GFP was excluded from the nucleus and appeared to be localized mainly at the PM, although some fluorescence was observed in intracellular vesicle-like compartments and in the cytoplasm. Phosphorylation assays indicated that SERK1 can autophosphorylate and transphosphorylate myelin basic protein (MPB). The autophosphorylation activity seemed enhanced when the kinase was mixed with the MBP substrate. SlSERK1 transcripts were detected in all tissues tested albeit with variable levels of expression. SlSERK1 is preferentially expressed in shoots, with the highest expression levels found in young leaves and flowers.
Publications
- Bhattarai, K. K., H. S. Atamian, I. Kaloshian*, and T. Eulgem*. 2010. WRKY72-type transcription factors contribute to basal immunity in tomato and Arabidopsis as well as gene-for-gene resistance mediated by the tomato R-gene Mi-1. Plant J 63:229-240 (* co-corresponding authors).
- Gerardo, N. N., B. Altincicek, C. Anselme, H. Atamian, and 22 additional authors including I. Kaloshian. 2010. Immunity and other defenses in pea aphids, Acyrthosiphon pisum. Genome Biology 11:R21.
- International Aphid Genomics Consortium (including H. Atamian and I. Kaloshian). 2010. Genome sequence of the pea aphid Acyrthosiphon pisum. PLoS Biology. Vol. 8: e1000313.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: The tomato (SOLANUM LYCOPERSICUM) gene Mi-1 is the only cloned resistance gene that confers resistance to root knot nematodes (RKN; MELOIDOGYNE spp.). In addition to RKN, MI-1 confers resistance to potato aphids, whiteflies and psyllids. The long-term goal of this project is to dissect the MI-1 defense signal transduction pathway. The specific objectives of this project are: 1) Evaluate the role of ET signaling pathway in Mi-1 resistance to aphids and RKNs 2) Evaluate the role members of MAP kinase pathways in Mi-1 resistance to RKNs 3) Identify additional components of MI-1 signal transduction pathway 4) Clone and characterize MI-9. We have reported earlier results for objective 1 and 2. In this period, progress accomplished under objectives 2 and 4 are presented. Results from this work were present at invited lecture/seminar at Gent University, Belgium, Wageningen University, the Netherlands as well as presented at the International Congress on Molecular Plant-Microbe Interactions in Quebec, Canada. PARTICIPANTS: Isgouhi Kaloshian- principle investigator, Hagop Atamian- graduate student, Kishor Bhattarai- graduate student, Olivia Desmond- postdoctoral fellow, Sophie Mantelin- postdoctoral fellow Tori Owens- Undergraduate student TARGET AUDIENCES: This work provided training to two graduate students and two postdoctoral fellows. Results were presented at the 14th International Congress on Molecular Plant-Microbe Interactions in Quebec, Canada, July 2009. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Tomato TOM1 cDNA microarray was used to identify transcriptional regulators involved in defense triggered by the Mi-1 protein. Near isogenic tomato cultivars resistant (Motelle) and susceptible (Moneymaker) to RKN were infected with an avirulent MELOIDOGYNE JAVANICA. Using linear model for microarray data analysis (Limma) differentially regulated genes in both susceptible and resistant tomato were identified. WRKY transcription factors differentially regulated in the resistant or incompatible interaction was further characterized for their role in plant defense. Using virus-induced gene silencing, two related transcription factors WRKY72a and WRKY72b were silenced in tomato and their roles in RKN and potato aphid resistance were evaluated. To further evaluate the role of these genes T-DNA insertion lines of Arabidopsis thaliana (Arabidopsis) for both genes were identified and used to evaluate resistance to bacteria PSEUDOMONAS SYRINGAE (PST), green peach aphid MYZUS PERSICAE, and the oomycetes HYALOPERONOSPORA ARABIDOPSIDIS (HPA). Expression of the two WRKY genes after RKN inoculation or aphid infestation were analyzed using real-time PCR. Microarray analysis was performed to profile transcriptional reprogramming associated with AtWRKY72-dependent basal defense to HPA strain Noco2 using Col-0 and both wrky72 mutants. A heat-stable resistance, mediated by the MI-9 gene, conferring 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-1.2 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. We screened an LA2157 gDNA Lambda library using the CC, NBS, or intron-1 as probes. Identified clones were 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. One of the LA2157 clones with homology to MI-1.1 and a predicted heat-stable motif was 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. In addition, LA2157 gDNA or cDNA has been used as a template for PCR-based cloning of Mi homologs. Clones were restriction digested for fingerprinting and categorized into groups and several representatives from each group were sequenced.
Publications
- 1. 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 Pathol. 57:1125-1135.
- 2. Mantelin, S., K. K. Bhattarai, and I. Kaloshian. 2009. Ethylene contributes to potato aphid susceptibility in a compatible tomato host. New Phytol. 183:444-456.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: In tomato, resistance to root-knot nematodes and potato aphids is conferred by the MI-1 gene that encodes a protein with nucleotide-binding site and leucine-rich repeat motifs. Several plant hormones play roles in plant defense against insects and pathogens. We assessed the role of the plant hormone ethylene (ET) in the interaction between resistant (incompatible) and susceptible (compatible) tomato and potato aphids. We concomitantly used genetic and pharmacologic approaches to impair ET production or perception in tomato. Genetic crosses were performed between cv. VFN (MI-1/MI-1 nr/nr) and the ET mutant Nr (mi/mi Nr/Nr) with a codominant mutation in the ET receptor 3 (ETR3) to introduce the MI-1 gene in the Nr mutant background. Homozygous (MI-1/MI-1 Nr/Nr) F2 and F3 plants insensitive to ET were selected for further studies. We also used tobacco rattle virus-based virus-induced gene silencing system to repress tomato ET receptor 4 (ETR4) and ET biosynthetic genes ACC synthase (ACS). To impair ET perception, tomato plants were treated for 24 h with SmartFresh (0.14% 1-methylcyclopropene [MCP]) in an airtight container. Resistant and susceptible tomato plants impaired in ET biosynthesis or perception were evaluated in choice or non-choice aphid bioassays. To measure ET production in tomato by aphid feeding, single tomato leaflets were placed in glass Mason jars with the cut stem inserted in 1x Murashige and Skoog supplemented with sucrose and agarose. Two hours later, leaflets were infested with potato aphids. Jars were covered with a double layer of cheesecloth and placed in the greenhouse inside a large plant cage. Infested and non-infested jars were sealed with a perforated lid containing a rubber syringe cap to allow collection of generated ET. After a 12 h incubation period, the headspace was sampled and the ET content was measured using a 6850 series gas chromatography system equipped with a HP Plot alumina-based capillary column. The expression of ET biosynthetic genes was evaluated by RT-PCR after aphid feeding. For the expression study, a time-course aphid infestation was performed with resistant and susceptible tomato plants. We constructed a genomic DNA library derived from leaf tissue of SOLANUM ARCANUM accession LA2157, the source of the heat-stable root-knot nematode resistance gene MI-9. LA2157 DNA was purified, partially restricted with SAU3A, ends were partially filled in, ligated into Lambda FIX II vector, packaged and propagated on XL1-Blue MRA (P2) E. COLI cells. Primary, secondary and tertiary screens of this phage library was performed using a probe (IK3-3), from the nucleotide-binding site of MI-1. PARTICIPANTS: Isgouhi Kaloshian- principle investigator, Kevin Izquierdo- undergraduate student, Kishor Bhattarai- graduate student, Olivia Desmond- postdoctoral fellow, Sophie Mantelin- postdoctoral fellow. TARGET AUDIENCES: This work provided training to one undergraduate student, one graduate student, and two postdoctoral fellows. Results were presented at the West Coast Biological Sciences Undergraduate Research Conference, April 12, 2008, San Diego, CA. Title of presentation "The role of ethylene in tomato plant immunity: Basal and resistance gene-mediated defense against potato aphids". Results were also presented at the International Nematology Congress in Brisbane, Australia, July 2008, and at a seminar in the Department of Nematology, UC Riverside. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
We demonstrated in this reporting period that ET biosynthetic genes were differentially regulated in resistant and susceptible tomato by aphid feeding. A burst of ET was detected in tomato during both compatible and incompatible interactions although the ET burst was delayed in the incompatible interaction. In addition, reducing ET sensitivity by attenuating the hormone perception or impairing ET biosynthesis decreased susceptibility during the compatible interaction but did not compromise MI-1-mediated resistance to the aphid. Our results indicate that ET may not play a significant role in MI-1-mediated resistance to potato aphids in tomato but modulates the host basal defense enhancing its susceptibility to the aphid. We developed a genomic DNA library from the wild tomato species SOLANUM ARCANUM accession LA2157. The primary library was tittered and found to contain 1.91x106 plaque forming units per mL. The insert size ranged between 10-23 Kb with an average insert size around 14 Kb. Screening of this library has identified 11 unique clones containing sequences with homology to the nematode resistance gene MI-1.
Publications
- Bhattarai, K. K., Q. Li, Y. Liu, S. P. Dinesh-Kumar, and I. Kaloshian. 2007. The Mi-1-mediated pest resistance requires Hsp90 and Sgt1. Plant Physiol. 144:312-323 Bhattarai, K., Q.-G. Xie, D. Pourshalimi, T. Younglove, and I. Kaloshian. 2007. LeCoi1-regulated JA-dependent signaling pathway is not required for the Mi-1-mediated potato aphid resistance. Mol. Plant-Microbe Interact. 20:276-282. 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.
- Bhattarai, K. K., Q-G. Xie, S. Mantlein, U. Bishnoi, T. Girke, D. A. Navarre, and I. Kaloshian. 2008. Tomato susceptibility to root-knot nematodes requires an intact JA signaling pathway. Mol. Plant-Microbe Interact. 21:1205-1214.
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Progress 01/01/05 to 12/31/05
Outputs
The tomato gene MI confers resistance against root-knot nematodes (MELOIDOGYNE spp.), potato aphid (MACROSIPHUM EUPHORBIAE) and whitefly (BEMISIA TABACI). MI was cloned and shown to belong to the largest class of resistance genes encoding proteins with nucleotide binding site and leucine-rich repeat motifs. Resistance gene-mediated responses involve global changes in gene expression mediated by multiple signaling pathways. These defense pathways are mediated by a number of small molecules including salicylic acid (SA), jasmonic acid (JA) and ethylene. We questioned whether SA and JA have a role in MI-1-mediated resistance to either nematodes or aphids. To address this question, we generated Mi-1 tomato lines with mutations in LeCOI-1 defective in JA signaling. We have also generated tomato with mutation in the SA signaling by introducing the NAHG transgene in MI-1 tomato. Screening MI tomato with the NAHG transgene indicated a role for SA in MI-mediated resistance to
aphid and nematodes. Currently, we are investigating a role for JA in MI-1-mediated resistance to both pests. Resistance signal pathways of diverse resistance gene seem to converge downstream of the R genes. Based on this observation, obvious candidates of the MI-1-mediated resistance signaling are genes involved in other R gene-mediated resistance signaling. To identify these candidate genes, we used virus-induced gene silencing (VIGS) to address the role of mitogen-activated protein kinases (MAPKs) in MI-1-mediated resistance to aphids. VIGS is a reverse genetic tool that allows rapid and transient suppression of host genes, by targeting sequence-specific degradation of their transcripts. Using VIGS in MI tomato, we identified a role for MAPK kinase (NtMEK2) and three MAPKs (SIPK, NTF4, and WIPK) in MI-1-mediated aphid resistance.
Impacts
MI-1 is the only plant gene that confers resistance to pests from distinct groups, aphids and nematodes. Understanding how MI-1 works, will lead to engineering broad-spectrum resistance.
Publications
- Kaloshian, I. and L. L. Walling. 2005. Hemipterans as Plant Pathogens. Annu. Rev. Phytopathol. 43:491-521.
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Progress 01/01/04 to 12/31/04
Outputs
Root-knot nematodes, MELOIDOGYNE SPP., are important agricultural pests worldwide that cause severe damage of many cultivated plant species including tomato, LYCOPERSICON ESCULENTUM. Currently, all commercially available root-knot nematode resistance in tomato cultivars is conferred by the single dominant gene MI. Over the years, virulent root-knot nematode isolates that are able to reproduce on MI-carrying tomato cultivars have been reported. In recent years, the emergence of two MI-virulent root-knot nematode populations, from different geographical locations in California, were also reported. Some of these MI-virulent isolates have been selected on MI-carrying tomato either by continuous propagation of avirulent isolates, or isolated from tomato roots from fields following repetitive planting of tomato cultivars carrying the MI gene. However, other isolates have been identified with inherent ability to parasitize tomato with MI. Additional sources of resistance to
MELOIDOGYNE INCOGNITA, M. JAVANICA, M. ARENARIA and M. HAPLA were identified in LYCOPERSICON PERUVIANUM Acc. PI 126443 clone 1MH, PI 270435 clones 3MH and 2R2. Each accession carried a different resistant gene, MI-3, MI-7 or MI-8, respectively. Unlike MI, the resistance in these accessions was heat-stable at 32 oC. We compared the reproduction of four geographically distinct MI-virulent root-knot nematode isolates on the three resistant accessions of LYCOPERSICON PERUVIANUM. All nematode isolates were verified as M. INCOGNITA using diagnostic markers in the mitochondrial genome of the nematode. Reproduction of MI-virulent isolates W1, 133 and HM, measured as eggs per g of root, was greatest on the MI-7 carrying accession and least on the MI-8 carrying accession. In general, MI-3 behaved similar to the MI-8 carrying accession. Reproduction of the four nematode isolates was also compared on both MI and non-MI-carrying L. ESCULENTUM cultivars and a susceptible L. PERUVIANUM accession.
Resistance mediated by MI in L. ESCULENTUM still impacted the MI-virulent nematodes with fewer eggs per g of root on the resistant cultivar. Preliminary histological studies suggests that MI-8 resistance is mediated by a hypersensitive response, similar to MI.
Impacts
Identifying new sources of resistance to MI-virulent nematode will assist in breeding new resistant tomato varieties. Pyramiding root-knot nematode resistance genes will assist in breeding durable resistant tomato cultivars.
Publications
- Huang, X., M. McGiffen and I. Kaloshian. 2004. Reproduction of MI-virulent MELOIDOGYNE INCOGNITA isolates on resistant and susceptible LYCOPERSICON SPP. J. Nematology. 36:69-75.
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Progress 01/01/03 to 12/31/03
Outputs
One of the goals of the research in my lab is to understanding how the tomato gene MI-1 confers resistance against 3 species of root-knot nematodes(MELOIDOGYNE SPP.) and to the potato aphid (MACROSIPHUM EUPHORBIAE). The MI-1 mediated nematode resistance is accompanied by cell death in the roots where the nematodes try to feed. We investigated whether cell death is also involved in the resistance mediated to aphids. Our results showed that no cell death occurs in the leaves after aphid feeding on resistant plants. In addition, we found reactive oxygen species generated by aphid feeding in both compatible and incompatible interactions. The induction of defense-related genes, locally and systemically, in response to potato aphid was investigated. Changes in mRNA levels of pathogenesis-related (PR) proteins, PR-1, GLUB (basic beta-1,3-glucanase), CHI3 (acidic chitinase) and proteinase inhibitors (PIN1 and PIN2) as well as LOXD (lipoxygenase) were assessed. Transcripts for
PR-1 and GLUB could be detected 12 h after infestation (hai) and reached a maximum at 48 hai. At this time, in tomato plants containing MI-1, systemic expression of PR-1 and GLUB was also detected. LOXD RNAs accumulated in both compatible and incompatible interactions. Similarly, in both types of interactions, PIN1 and PIN2 transcripts were detected at 6 and 12 hai only.
Impacts
Root-knot nematodes and aphids are serious pests of tomato. Understanding how resistant gene work will enable us to engineer durable resistance.
Publications
- Martinez de Ilarduya, O., Q.-G. Xie, and I. Kaloshian. 2003. Aphid-induced defense responses in MI-1-mediated compatible and incompatible tomato interactions. Mol. Plant-Microbe Interact. 16:699-708 (Featured on the cover of this issue).
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Progress 01/01/02 to 12/31/02
Outputs
We continue our work towards understanding how the tomato gene MI-1 confers resistance against 3 species of root-knot nematodes(MELOIDOGYNE SPP.) and to the potato aphid (MACROSIPHUM EUPHORBIAE). We have identified a new member in the Mi-mediated pathway to both organisms. In addition, we have fine-mapped a novel root-knot nematode resistance gene to the short arm of chromosome 6 of tomato. While the MI-1 mediated resistance is heat-sensitive (breaks down above 28 C), the resistance mediated by the novel gene is heat stable (at 32 C).
Impacts
Nematodes are serious pest of tomato. Identifying and characterizing novel sources of resistance is crucial for the tomato industry. Understanding how resistant gene work will enable us to engineer durable resistance.
Publications
- Kaloshian, I. and O. Martinez de Ilarduya. 2002. The tomato RME1gene is required for Mi-1-mediated resistance. 208-211. In: Biology of Plant-Microbe Interactions. Vol III. Proceedings of 10th International Congress, Plant-Microbe Interactions, Madison, Wisconsin, Leong, S. A., C. Allen, and E.W. Triplett, Eds. International Society of Mol. Plant-Microbe Interactions, St. Paul, Minnesota, USA.
- Huang, X., P. S. Springer and I. Kaloshian. 2003. Expression of the Arabidopsis MCM gene PROLIFERA during root-knot and cyst nematode infection. Phytopathology 93:35-41.
- Bird, D. Mck. and I. Kaloshian. (2003) Are roots special? Nematodes have their say. Physiological Molecular Plant Pathology (in press).
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Progress 01/01/01 to 12/31/01
Outputs
The tomato gene MI is the only commercially available resistance gene that confers resistance against 3 species of root-knot nematodes, MELOIDOGYNE spp. MI also confers resistance to the potato aphid (MACROSIPHUM EUPHORBIAE). MI was cloned and shown to belong to a large class of resistance genes with predicted leucine zipper, nucleotide binding site and leucine rich repeats. This indicates that MI-mediated resistance most likely requires the presence of other genes or factors to succeed. We are using a genetic approach to identify suppressors of MI-mediated resistance. We have identified several mutants that allow different levels of nematode reproduction in the presence of a functional MI. One of these mutants rme1, shows complete susceptibility to both nematodes and aphid. To determine whether RME1 functions in a general disease-resistance pathway, the response against FUSARIUM OXYSPORUM f.sp. LYCOPERSICI race 2, mediated by the I-2 resistance gene, was studied.
Both rme1 and the wild type plants were equally resistant to the fungal pathogen. These results indicate that RME1 does not play a general role in disease resistance but may be specific for MI-1-mediated resistance. In conjunction to understanding how MI-1 mediates resistance, we are also mapping novel sources of nematode resistance in LYCOPERSCON PERUVIANUM accessions LA2157 and PI 270435-2R2. LA2157 confers heat-stable resistance to MI-aviruelnt nematodes while PI 270435-2R2 confers both heat-stable resistance and resistance to MI-virulent nematodes. Using both RFLP and PCR-based markers with 216 F2 individuals, we have mapped MI-9 to a small genetic interval between DNA markers CT119 and C8B on the short arm of chromosome 6. As for mapping the heat-stable resistance in PI 270435-2R2, BSA and AFLP were performed by pooling DNAs from 10 resistant and 10 susceptible individuals. A total of 128 primer combinations were used, which revealed 30 polymorphic markers. Linkage analysis with
the whole population identified one marker to be tightly linked in repulsion-phase and another marker in coupling-phase, with a distance of 16 cM from the gene. Work is in progress to clone these AFLP markers for mapping. Resistance to MI-1-viruent nematode was also evaluated with this population. 92 pseudo backcross progeny were screened with MI-1-virulent M. INCOGNITA at 25C. Genetic analysis revealed the presence of 26 resistant and 66 susceptible individuals 1:3 (X2 = 0.016 P <0.05) suggesting that the resistance to virulent nematode may be governed by more than one gene. Roots were air dried and weighed. We observed a large variation in the number of egg masses per gram of root. These finding suggest that, resistance to virulent RKN may be quantitatively inherited. Therefore, quantitative trait loci mapping approach will be undertaken to identify loci controlling resistance and the phenotypic contribution of each locus to the trait.
Impacts
Root-knot nematodes are serious pest of tomato. Identifying and characterizing novel sources of resistance is crucial for the tomato industry. Understanding how resistant genes work will enable us to engineer durable resistance.
Publications
- Ammiraju, J. S. S., J. C. Veremis, X. Huang, P. A. Roberts, I. Kaloshian. 2002. Fine mapping of the heat-stable root-knot nematode resistance gene MI-9 in LYCOPERSICON PERUVIANUM. Theoretical and Applied Genetics (In press).
- Martinez de Ilarduya, O., A. E. Moore and I. Kaloshian. 2001. The tomato Rme1 locus is required for Mi-1-mediated resistance to root-knot nematodes and the potato aphid. Plant J. 27:417-425.
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Progress 01/02/00 to 12/31/00
Outputs
The tomato gene, MI-1, is the first cloned plant resistance gene with dual specificity to two distinct organisms, nematodes and an aphid. Despite this uniqueness, MI-1 encodes a protein sharing molecular features with a number of single-specificity resistance genes. Two strategies are being utilized in my laboratory to identify plant genes that control resistance to root-knot nematodes (RKNs). The first is to dissect genetically the MI-1 mediated resistance pathway to both organisms. We are using mutational analysis for identification and characterization of tomato genes, other than Mi-1, that control nematode and aphid recognition and the subsequent expression of resistance. We have identified a mutant that is compromised in both nematode and aphid resistances. This mutant is not altered in resistances mediated by other resistance genes in this tomato cultivar. This indicates that we have identified a member of the MI-mediated resistance signal transduction pathway.
The second approach is to clone resistance genes to RKNs with novel specificities. Currently, only one nematode resistance gene, MI-1, is present in cultivated tomato. The value of MI-1 has been compromised partially by the appearance at several locations in California and in other parts of the world, of RKN variants that can infect MI-1-bearing tomato. Soil temperatures above 28C also inactivate MI-1 effectiveness. Several new resistance genes to RKNs have been identified in the wild tomato species, LYCOPERSICON PERUVINUM. These novel genes either confer resistance to RKN isolates that can parasitize tomato with MI-1 or express resistance at high temperatures. We are using map-based cloning approach to clone these resistance genes. We have developed segregation populations of L. PERUVINUM and have screened them with RKNs. Currently, we are using Amplified Fragment Length Polymorphism analysis to identify linked markers to these traits.
Impacts
Because of environmental impact of nematicides and fumigants, plant disease resistance is increasingly the method of choice to control nematodes. Cloning novel resistance genes and understanding how these genes mediate resistance will enable us to engineer durable resistance.
Publications
- Kaloshian, I. and O. Martinez de Ilarduya 1999. MI-1, a dual function disease resistance gene in tomato. In: Delivery of Pathogen Signals to Plants. Proc. 8th Japan-U.S. Seminar, Keen, N. T. and S. Mayama, Eds. APS Press, St Paul, MN. O. Martinez de Ilarduya and I. Kaloshian. 2001. MI-1.2 transcripts accumulate ubiquitously in resistant LYCOPERSICON ESCULENTUM. Journal of Nematology (In Press)
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Progress 01/01/99 to 12/31/99
Outputs
We have taken the first steps towards mapping of nematode resistance gene clusters in wild tomato LYCOPERSICON PERUVIANUM. We are using 2 different L. PERUVIANUM lines, carrying 2 different nematode resistance genes each. We have generated 2 segregating population of L. PERUVIANUM, each for one pair of the resistance clusters. A subset of these populations has been planted and a number of cuttings have been made from individual plants. Since L. PERUVIANUM is an outcrosser, in order to obtain a number of plants from the same genotype, cuttings are made from the individual F2 plants. These cuttings are used in duplicate in every nematode screen. Cuttings, generated from the same plant, have been screened with nematode isolate W-1 (MI -virulent MELOIDOGYNE INCOGNITA and isolate VW-4 (regular M. JAVANICA) at room temperature and 88 F, respectively. We have evaluated most of the initial populations (total of 140 plants), in 4 separate experiments, for each nematode. This
is done to make sure that the phenotype of the plants is correctly identified. The results of these screens, confirmed earlier findings that these genes are inherited in a dominant manner. At present we are using these segregating populations to identify molecular markers linked to these resistance genes. Besides mapping novel resistance clusters in tomato, our lab is also interested in understanding how the tomato resistance gene, MI-1, confers resistance to nematodes. Towards this goal we have generated 4 mutagenized populations of tomato containing the MI-1 gene. These tomato populations are mutagenized with 2 different mutagens, EMS and fast neutron. We are screening for nematode susceptibility or reduced resistance in the presence of MI-1 gene. We have screen over 800 M2 families with root-knot nematodes. Briefly, 25 M2 seeds, collected from single M2 plants, were individually inoculated with nematodes. Six to eight weeks after inoculation, plants were checked for nematode
infections, measured as number of egg masses developed on the root system. We have identified four confirmed mutants, showing different degrees of infection. One mutant, RME1 (for resistance to MELOIDOGYNE) isolated from fast neutron irradiated seeds, showed complete susceptibility. The other three mutants (RME2, RME3, and RME4), showed intermediate or reduced nematode resistance. We are in the process of characterizing these mutants.
Impacts
Cloning novel resistance genes and understanding how resistance genes confer resistance to nematodes will allow us to engineer durable resistance to these agronomically important pathogens.
Publications
- No publications reported this period
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