The Mi-1 gene of tomato confers resistance against three species of root-knot nematode in tomato (Lycopersicon esculentum). Transformation of tomato carrying Mi-1 with a construct expressing NahG, which encodes salicylate hydroxylase, a bacterial enzyme that degrades salicylic acid (SA) to catechol, results in partial loss of resistance to root-knot nematodes. Exogenous SA was toxic to roots expressing NahG but not to control roots. This toxicity is most likely due to the production of catechol from SA, and we report here that 100 microM catechol is toxic to tomato roots. Benzothiadiazole, a SA analog, completely restores nematode resistance in Mi-1 roots transformed with NahG but does not confer resistance to susceptible tomato roots. The localized cell death produced by transient expression in Nicotiana benthamiana of Mi-DS4, a constitutively lethal chimera of Mi-1 with one of its homologs, was prevented by coexpression of NahG. These results indicate that SA is an important component of the signaling that leads to nematode resistance and the associated hypersensitive response.

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.

SummaryThe 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.

Summary In tobacco and other Solanaceae species, the tobacco N gene confers resistance to tobacco mosaic virus (TMV), and leads to induction of standard defense and resistance responses. Here, we report the use of N‐transgenic tomato to identify a fast‐neutron mutant, sun1‐1 (suppressor of N), that is defective in N‐mediated resistance. Induction of salicylic acid (SA) and expression of pathogenesis‐related (PR) genes, each signatures of systemic acquired resistance, are both dramatically suppressed in sun1‐1 plants after TMV treatment compared to wild‐type plants. Application of exogenous SA restores PR gene expression, indicating that SUN1 acts upstream of SA. Upon challenge with additional pathogens, we found that the sun1‐1 mutation impairs resistance mediated by certain resistance (R) genes, (Bs4, I, and Ve), but not others (Mi‐1). In addition, sun1‐1 plants exhibit enhanced susceptibility to TMV, as well as to virulent pathogens. sun1‐1 has been identified as an EDS1 homolog present on chromosome 6 of tomato. The discovery of enhanced susceptibility in the sun1‐1 (Le_eds1‐1) mutant plant, which contrasts to reports in Nicotiana benthamiana using virus‐induced gene silencing, provides evidence that the intersection of R gene‐mediated pathways with general resistance pathways is conserved in a Solanaceous species. In tomato, EDS1 is important for mediating resistance to a broad range of pathogens (viral, bacterial, and fungal pathogens), yet shows specificity in the class of R genes that it affects (TIR‐NBS‐LRR as opposed to CC‐NBS‐LRR). In addition, a requirement for EDS1 for Ve‐mediated resistance in tomato exposes that the receptor‐like R gene class may also require EDS1.

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. INTRODUCTIONDisease resistance in plants is often characterized by a gene-for-gene relationship that requires a specific plant resistance ( R ) gene and a corresponding pathogen avirulence ( avr ) gene (Flor, 1955). Recognition initiates a cascade of defense responses, often including a hypersensitive response (HR) consisting of localized cell death at the infection site. Recently, R genes that mediate resistance to viruses, bacteria, fungi, and nematodes have been cloned from several plant species (Baker et al., 1997;Hammond-Kosack and Jones, 1997). Most encode proteins that carry a structural motif with a repeating pattern of 20 to 30 amino acids called a leucine-rich repeat (LRR). LRR-containing R genes can be subdivided into two broad classes: those in which the predicted gene product contains an N-terminal, extracellular LRR and a membrane anchor; and those in which the R gene product is predicted to be cytoplasmic. Cytoplasmically located R gene products are characterized by the presence of a conserved region containing a nucleotide binding site (NBS) and a C-terminal LRR region.Root-knot nematodes (genus Meloidogyne ) are parasitic roundworms that damage Ͼ 1000 different food and fiber crops around the world (Williamson and Hussey, 1996). In a compatible interaction, second-stage nematode juveniles (J2s) penetrate the host, generally near root tips, then migrate intercellularly to the region of cell differentiation. In response to signals from the nematode, plant cells adjacent to the head of the nematode enlarge to form large, multinucleate, metabolically active cells that serve as the source of nutrients for the developing, endoparasitic form of the nematode. Concurrent hyperplasia and hypertrophy in the surrounding host tissues lead to the formation of the galls or root knots characteristic of Meloidogyne spp infection.Tomato is an excellent host for root-knot nematodes. Howev...

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Nitrate redudase (NR) is the first enzyme in nitrate assimilation, a critica1 process for plant survival. l h e regulation of NR gene expression is complex, involving both interna1 and externa1 factors. O f these, nitrate indudion of NR gene expression has been studied most extensively and is well conserved among baderia, fungi, and higher plants. We are interested in understanding the mechanism of nitrate indudion of higher plant NR genes. Here we describe promoter analyses of the 5' flanking regions of the Arabidopsis NR genes, NR1 and NRZ, with resped to nitrate indudion of gene expression. To facilitate these analyses, a nitrate indudion procedure using 1, transgenic tobacco plants was established. Approximately 1.5-kb 5' flanking regions of the two Arabidopsis NR genes (NR1 and NRZ) were fused to a reporter gene and its expression in transgenic plants was analyzed. Deletion analyses of these regions show that 238-and 188-bp 5' flanking regions of the NR1 and NRZ, respedively, contain sequences responsive to nitrate indudion.

Here we identify the cis-acting elements of NP1 and NP2 that are necessary for nitrate-dependent transcription by linker-scanning (1s) analysis. In transgenic plants one LS mutant of NP1 and two LS mutants of NP2 exhibited significantly lower nitrate-induced reporter gene chloramphenicol acetyltransferase activity. To distinguish which of these three mutants lost nitrate inducibility, competitive reverse-transcriptase polymerase chain reaction was used t o measure the chloramphenicol acetyltransferase mRNA levels before and after nitrate induction. The single LS mutant in N P l lost its response to nitrate, whereas the t w o LS mutants in NP2 partially lost their response to nitrate. A 12-bp sequence is conserved between the NP1 site and the two NP2 sites. This sequence motif is also conserved i n the 5' flanking regions of other nitrate-inducible plant genes. Cel mobility shift experiments indicate that these three regions bind t o similar proteins. The binding is constitutive with respect t o nitrate treatment and was observed in both nonphotosynthetic suspension cells and green leaves.