Mi-1-Mediated aphid resistance involves salicylic acid and mitogen-activated protein kinase signaling cascades

The tomato Mi-1 gene confers resistance to root-knot nematodes (Meloidogyne spp.), potato aphids (Macrosiphum eluphorbiae), and whiteflies (Bemisia tabaci and B. tabaci biotype B). Resistance to potato aphid is developmentally regulated and is not associated with induction of a hypersensitive response. The NahG transgene that eliminates endogenous salicylic acid (SA) was used to test the role of the SA signaling pathway in the resistance mediated by Mi-1 to potato aphids. Aphids survived longer on NahG tomato plants than on wild type. However, aphid reproduction was not affected on NahG tomato. Aphid resistance in Mi-1 NahG plants was completely abolished and the phenotype was successfully rescued by application of BTH (benzo(1,2,3)-thiaiazole-7-carbothioic acid S-methyl ester), indicating that the SA signaling pathway is an important component of Mi-1-mediated aphid resistance. Using virus-induced gene silencing, one or more mitogen-activated protein kinase (MAPK) cascades required for Mi-1-mediated aphid resistance were identified. Silencing plants for MAPK kinase (LeMKK2) and MAPKs (LeMPK2 and LeMPK1, or LeMPK3) resulted in attenuation of Mi-1-mediated aphid resistance. These results further demonstrate that resistance gene-mediated signaling events against piercing-sucking insects are similar to those against other plant pathogens.     

The tomato Rme1 locus is required for Mi-1-mediated resistance to root-knot nematodes and the potato aphid

The tomato Mi-1 gene confers resistance against root-knot nematodes (Meloidogyne spp.) and a biotype of the potato aphid (Macrosiphum euphorbiae). Four mutagenized Mi-1/Mi-1 tomato populations were generated and screened for altered root-knot nematode resistance. Four independent mutants belonging to two phenotypic classes were isolated. One mutant was chosen for further analyzes; rme1 (for resistance to Meloidogyne) exhibited levels of infection comparable with those found on susceptible controls. Molecular and genetic data confirmed that rme1 has a single recessive mutation in a locus different from Mi-1. Cross-sections through galls formed by feeding nematodes on rme1 roots were identical to sections from galls of susceptible tomato roots. In addition to nematode susceptibility, infestation of rme1 plants with the potato aphid showed that this mutation also abolished aphid resistance. 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.     

Heterologous expression of the Mi-1.2 gene from tomato confers resistance against nematodes but not aphids in eggplant

The Mi-1.2 gene in tomato (Solanum lycopersicum) is a member of the nucleotide-binding leucine-rich repeat (NBLRR) class of plant resistance genes, and confers resistance against root-knot nematodes (Meloidogyne spp.), the potato aphid (Macrosiphum euphorbiae), and the sweet potato whitefly (Bemisia tabaci). Mi-1.2 mediates a rapid local defensive response at the site of infection, although the signaling and defensive pathways required for resistance are largely unknown. In this study, eggplant (S. melongena) was transformed with Mi-1.2 to determine whether this gene can function in a genetic background other than tomato. Eggplants that carried Mi-1.2 displayed resistance to the root-knot nematode Meloidogyne javanica but were fully susceptible to the potato aphid, whereas a susceptible tomato line transformed with the same transgene was resistant to nematodes and aphids. This study shows that Mi-1.2 can confer nematode resistance in another Solanaceous species. It also indicates that the requirements for Mi-mediated aphid and nematode resistance differ. Potentially, aphid resistance requires additional genes that are not conserved between tomato and eggplant.     

Leucine-rich repeat-mediated intramolecular interactions in nematode recognition and cell death signaling by the tomato resistance protein Mi

The root-knot nematode resistance gene Mi from tomato encodes a nucleotide-binding/leucine-rich repeat (NB/LRR) protein with a novel amino-terminal domain compared to related disease-resistance genes. The closely linked paralog Mi-1.1, which does not confer nematode resistance, encodes a protein 91% identical to the functional copy, Mi-1.2. The chimeric construct Mi-DS3, which encodes the 161 amino-terminal residues from Mi-1.1 fused to the remainder of Mi-1.2, induces localized necrosis when transiently expressed in Nicotiana benthamiana leaves. We produced mutant constructs that exchanged sequences encoding each of the 40 amino acid differences from the Mi-1.1 LRR region into Mi-DS3 and into Mi-1.2. For 23 of the substitutions, necrosis was lost upon transient expression of the mutated Mi-DS3 in N. benthamiana, and nematode resistance was lost when the altered Mi-1.2 was expressed in the tomato roots. One substitution, R961D, failed to give Mi-DS3-induced necrosis, but produced a dominant lethal phenotype when introduced into Mi-1.2. This gain-of-function phenotype was suppressed by co-expression with the amino-terminal region of Mi-1.1, suggesting that residue 961 is critical for negative regulation by the corresponding N-terminal region. Substitutions of Mi-1.1 residues 984-986 retained the ability to cause necrosis in Mi-DS3, but resulted in loss-of-nematode resistance in Mi-1.2, suggesting that these residues are essential for nematode recognition. None of the loss-of-function mutations in Mi-1.2 had a dominant negative phenotype. These results indicate that the Mi-1.2 LRR is involved in regulation of the transmission of the resistance response as well as in recognition of the nematode.     

Loss of non-host resistance of Arabidopsis NahG to Pseudomonas syringae pv. phaseolicola is due to degradation products of salicylic acid

In plants carrying the NahG transgene, salicylate hydroxylase converts salicylic acid (SA) to catechol. Arabidopsis NahG plants are defective in non-host resistance to Pseudomonas syringae pv. phaseolicola strain 3121 (Psp), suggesting that resistance requires SA signaling. However, several mutants with defects in SA signaling, including eds1, pad4, eds5, sid2, and npr1, remain resistant to Psp, demonstrating that susceptibility of NahG plants is not due to absence of SA. SA synthesis is blocked in sid2NahG double mutants, but resistance to Psp is retained. Therefore, it must be the degradative action of NAHG on SA that causes the loss of resistance of NahG to Psp. Treatment of plants with catechol compromised Psp resistance suggesting that the effect of NahG on resistance results from catechol production. Application of catalase to NahG or catechol-treated wild-type plants partially restored resistance to Psp, suggesting that the deleterious effect of catechol results from inappropriate production of hydrogen peroxide. These results indicate that conclusions about SA requirements based solely on phenotypes of NahG plants should be re-evaluated.     

The Effects of Root-knot Nematode Infection and Mi-mediated Nematode Resistance in Tomato on Plant Fitness

The Mi-1.2 resistance gene in tomato (Solanum lycopersicum) confers resistance against several species of root-knot nematodes (Meloidogyne spp.). This study examined the impact of M. javanica on the reproductive fitness of near-isogenic tomato cultivars with and without Mi-1.2 under field and greenhouse conditions. Surprisingly, neither nematode inoculation or host plant resistance impacted the yield of mature fruits in field microplots (inoculum=8,000 eggs/plant), or fruit or seed production in a follow-up greenhouse bioassay conducted with a higher inoculum level (20,000 eggs/plant). However, under heavy nematode pressure (200,000 eggs/plant), greenhouse-grown plants carrying Mi-1.2 had more than ten-fold greater fruit production than susceptible plants and nearly forty-fold greater estimated lifetime seed production, confirming prior reports of the benefits of Mi-1.2. In all cases Mi-mediated resistance significantly reduced nematode reproduction. These results indicated that tomato can utilize tolerance mechanisms to compensate for moderate levels of nematode infection, but that the Mi-1.2 resistance gene confers a dramatic fitness benefit under heavy nematode pressure. No significant cost of resistance was detected in the absence of nematode infection.     

The MI-1-mediated pest resistance requires Hsp90 and Sgt1

The tomato (Solanum lycopersicum) Mi-1 gene encodes a protein with putative coiled-coil nucleotide-binding site and leucine-rich repeat motifs. Mi-1 confers resistance to root-knot nematodes (Meloidogyne spp.), potato aphids (Macrosiphum euphorbiae), and sweet potato whitefly (Bemisia tabaci). To identify genes required in the Mi-1-mediated resistance to nematodes and aphids, we used tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) to repress candidate genes and assay for nematode and aphid resistance. We targeted Sgt1 (suppressor of G-two allele of Skp1), Rar1 (required for Mla12 resistance), and Hsp90 (heat shock protein 90), which are known to participate early in resistance gene signaling pathways. Two Arabidopsis (Arabidopsis thaliana) Sgt1 genes exist and one has been implicated in disease resistance. Thus far the sequence of only one Sgt1 ortholog is known in tomato. To design gene-specific VIGS constructs, we cloned a second tomato Sgt1 gene, Sgt1-2. The gene-specific VIGS construct TRV-SlSgt1-1 resulted in lethality, while silencing Sgt1-2 using TRV-SlSgt1-2 did not result in lethal phenotype. Aphid and root-knot nematode assays of Sgt1-2-silenced plants indicated no role for Sgt1-2 in Mi-1-mediated resistance. A Nicotiana benthamiana Sgt1 VIGS construct silencing both Sgt1-1 and Sgt1-2 yielded live plants and identified a role for Sgt1 in Mi-1-mediated aphid resistance. Silencing of Rar1 did not affect Mi-1-mediated nematode and aphid resistance and demonstrated that Rar1 is not required for Mi-1 resistance. Silencing Hsp90-1 resulted in attenuation of Mi-1-mediated aphid and nematode resistance and indicated a role for Hsp90-1. The requirement for Sgt1 and Hsp90-1 in Mi-1-mediated resistance provides further evidence for common components in early resistance gene defense signaling against diverse pathogens and pests.     

Evidence for a role of the N terminus and leucine-rich repeat region of the Mi gene product in regulation of localized cell death

The tomato Mi gene confers resistance against root-knot nematodes and potato aphids. Chimeric constructs of the functional gene, Mi-1. 2, with a homolog, Mi-1.1, were produced, and their phenotypes were examined in Agrobacterium rhizogenes-transformed roots. Exchange of the leucine-rich repeat (LRR) region of Mi-1.1 into Mi-1.2 resulted in the loss of ability to confer nematode resistance, as did substitution of a 6-amino acid sequence from the Mi-1.1 LRR into Mi-1.2. Introduction of the Mi-1.2 LRR-encoding region into Mi-1.1 resulted in a lethal phenotype, as did substitution of the fragment encoding the N-terminal 161 amino acids of Mi-1.1 into Mi-1.2. Transient expression of the latter two chimeric constructs in Nicotiana benthamiana leaves produced localized cell death. The cell death caused by the N-terminal exchange was suppressed by coinfiltration with a construct expressing the N-terminal 161 amino acids of Mi-1.2. The phenotypes of these and other constructs indicate that the LRR region of Mi-1.2 has a role in signaling localized cell death and that the N-terminal 161 amino acids have a role in regulating this death.     

Effects of the Mi-1 and the N root-knot nematode-resistance gene on infection and reproduction of Meloidogyne enterolobii on tomato and pepper cultivars

Meloidogyne enterolobii is widely considered to be an aggressive root-knot nematode species that is able to reproduce on root-knot nematode-resistant tomato and pepper cultivars. In greenhouse experiments, M. enterolobii isolates 1 and 2 from Switzerland were able to reproduce on tomato cultivars carrying the Mi-1 resistance gene as well as an N-carrying pepper cultivar. Reproduction factors (Rf) ranged between 12 and 109 depending on the plant cultivar, with M. enterolobii isolate 2 being more virulent when compared to isolate 1. In contrast, M. arenaria completely failed to reproduce on these resistant tomato and pepper cultivars. Although some variability in virulence and effectiveness of root-knot nematode-resistance genes was detected, none of the plant cultivars showed Rf values less than 1 or less than 10% of the reproduction observed on the susceptible cv. 'Moneymaker' (Rf = 23-44) used to characterize resistance. The ability of M. enterolobii to overcome the resistance of tomato and pepper carrying the Mi-1 and the N gene makes it difficult to manage this root-knot nematode species, particularly in organic farming systems where chemical control is not an option.     

Salicylic acid is not required for Cf-2- and Cf-9-dependent resistance of tomato to Cladosporium fulvum

Tomato leaves or cotyledons expressing the Cf-2 or Cf-9 Cladosporium fulvum resistance genes induce salicylic acid (SA) synthesis following infiltration with intercellular washing fluid (IF) containing the fungal peptide elicitors Avr2 and Avr9. We investigated whether SA was required for Cf gene-dependent resistance. Tomato plants expressing the bacterial gene nahG, encoding salicylate hydroxylase, did not accumulate SA in response to IF infiltration but remained fully resistant to C. fulvum. NahG Cf0 plants were as susceptible to C. fulvum as wild-type Cf0. Neither free nor conjugated salicylic acid accumulated in IF-infiltrated Cf2 and Cf9 NahG leaves and cotyledons but conjugated catechol did accumulate. The Cf-9-dependent necrotic response to IF was prevented in NahG plants and replaced by a chlorotic Cf-2-like response. SA also potentiated Cf-9-mediated necrosis in IF-infiltrated wild-type leaves. In contrast, the Cf-2-dependent IF response was retained in NahG leaves and chlorosis was more pronounced than in the wild-type. The distribution of cell death between different cell types was altered in both Cf2 and Cf9 NahG leaves after IF injection. IF-induced accumulation of three SA-inducible defence-related genes was delayed and reduced but not abolished in NahG Cf2 and Cf9 leaves and cotyledons. NahG Tm-22 tomato showed increased hypersensitive response (HR) lesion size upon TMV infection, as observed in TMV-inoculated N gene-containing NahG tobacco plants.     

Mi-1-Mediated Nematode Resistance in Tomatoes is Broken by Short-Term Heat Stress but Recovers Over Time

Tomato (Solanum lycopersicum L.) is among the most valuable agricultural products, but Meloidogyne spp. (root-knot nematode) infestations result in serious crop losses. In tomato, resistance to root-knot nematodes is controlled by the gene Mi-1, but heat stress interferes with Mi-1-associated resistance. Inconsistent results in published field and greenhouse experiments led us to test the effect of short-term midday heat stress on tomato susceptibility to Meloidogyne incognita race 1. Under controlled day/night temperatures of 25°C/21°C, ‘Amelia’, which was verified as possessing the Mi-1 gene, was deemed resistant (4.1 ± 0.4 galls/plant) and Rutgers, which does not possess the Mi-1 gene, was susceptible (132 ± 9.9 galls/plant) to M. incognita infection. Exposure to a single 3 hr heat spike of 35°C was sufficient to increase the susceptibility of ‘Amelia’ but did not affect Rutgers. Despite this change in resistance, Mi-1 gene expression was not affected by heat treatment, or nematode infection. The heat-induced breakdown of Mi-1 resistance in ‘Amelia’ did recover with time regardless of additional heat exposures and M. incognita infection. These findings would aid in the development of management strategies to protect the tomato crop at times of heightened M. incognita susceptibility.     

The heat-stable root-knot nematode resistance gene <Emphasis Type="Italic">Mi-9</Emphasis> from Lycopersicon peruvianum is localized on the short arm of chromosome 6

The tomato gene Mi-1 confers resistance to three species of root-knot nematodes, Meloidogyne spp. However, the resistance mediated by Mi-1 is inactive at soil temperatures above 28 degrees C. Previously, we identified and mapped a novel heat-stable nematode resistance gene from the wild species Lycopersicon peruvianum accession LA2157 on to chromosome 6. Here we report further characterization of this heat-stable resistance against three Mi-1-avirulent biotypes of Meloidogyne javanica, Meloidogyne arenaria and Meloidogyne incognita. Screening segregating F(2) and F(3) progenies, derived from an intraspecific cross between susceptible LA392 and resistant LA2157, for nematode resistance at 25 degrees C and 32 degrees C, revealed a simple dominant monogenic inheritance with all the biotypes tested. We designate this gene as Mi-9. As a first step towards cloning of Mi-9, we constructed a linkage map around this gene. A total of 216 F(2) progeny from the cross between LA392 and LA2157 were screened with M. javanica at 32 degrees C and with CT119 and Aps-1, markers that flank the genetic interval that contains the Mi-1 gene. DNA marker analysis indicated that these markers also flank Mi-9. Further mapping of recombinants with both RFLP and PCR-based markers localized Mi-9 to the short arm of chromosome 6 and within the same genetic interval that spans the Mi-1 region.     

The Participation of salicylic and jasmonic acids in genetic and induced resistance of tomato to Meloidogyne incognita (Kofoid and White, 1919)

Salicylic (SA) and jasmonic (JA) acids are the best known mediators of signal systems in plants. In this investigation the participation and character of interactions between SA- and JA-signals under the induced and genetic resistance of plants to nematodes was investigated on the model system tomato (Lycopersicon esculentum) and the root-knot nematode Meloidogyne incognita. This study demonstrates that application of JA and SA to tomato foliage induces systemic effects that suppress root-knot nematode infestation, inhibition of nematode reproduction, and also increased activity of LOX and PAL, the enzymes of biosynthesis of JA and SA. JA treatment did not inhibit Mz-mediated resistance, which suggests a lack of signaling conflicts between these two forms of defense.     

The root-knot nematode resistance gene Mi-1.2 of tomato is responsible for resistance against the whitefly Bemisia tabaci

The tomato gene Mi-1.2 confers resistance against root-knot nematodes and some isolates of potato aphid. Resistance to the whitefly Bemisia tabaci previously has been observed in Mi-bearing commercial tomato cultivars, suggesting that Mi, or a closely linked gene, is responsible for the resistance. The response of two biotypes of B. tabaci to tomato carrying the cloned Mi was compared with that of the isogenic untransformed tomato line Moneymaker. Our results indicate that Mi-1.2 is responsible for the resistance in tomato plants to both B- and Q- biotypes. Mi-1.2 is unique among characterized resistance genes in its activity against three very different organisms (root-knot nematodes, aphids, and whiteflies). These pests are among the most important on tomato crops worldwide, making Mi a valuable resource in integrated pest management programs.     

Mapping a new nematode resistance locus in Lycopersicon peruvianum

Accessions of the wild tomato species L. peruvianum were screened with a root-knot nematode population (557R) which infects tomato plants carrying the nematode resistance gene Mi. Several accessions were found to carry resistance to 557R. A L. peruvianum backcross population segregating for resistance to 557R was produced. The segregation ratio of resistant to susceptible plants suggested that a single, dominant gene was a major factor in the new resistance. This gene, which we have designated Mi-3, confers resistance against nematode strains that can infect plants carrying Mi. Mi-3, or a closely linked gene, also confers resistance to nematodes at 32°C, a temperature at which Mi is not effective. Bulked-segregant analysis with resistant and susceptible DNA pools was employed to identify RAPD markers linked to this gene. Five-hundred-and-twenty oligonucleotide primers were screened and two markers linked to the new resistance gene were identified. One of the linked markers (NR14) was mapped to chromosome 12 of tomato in an L. esculentum/L. pennellii mapping population. Linkage of NR14 and Mi-3 with RFLP markers known to map on the short arm of chromosome 12 was confirmed by Southern analysis in the population segregating for Mi-3. We have positioned Mi-3 near RFLP marker TG180 which maps to the telomeric region of the short arm of chromosome 12 in tomato.