Kishor K. Bhattarai

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.

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

WRKY transcription factors play a central role in transcriptional reprogramming associated with plant immune responses. However, due to functional redundancy, typically the contribution of individual members of this family to immunity is only subtle. Using microarray analysis, we found that the paralogous tomato WRKY genes SlWRKY72a and b are transcriptionally up-regulated during disease resistance mediated by the R gene Mi-1. Virus-induced gene silencing of these two genes in tomato resulted in a clear reduction of Mi-1-mediated resistance as well as basal defense against root-knot nematodes (RKN) and potato aphids. Using Arabidopsis T-DNA insertion mutants, we found that their Arabidopsis ortholog, AtWRKY72, is also required for full basal defense against RKN as well as to the oomycete Hyaloperonospora arabidopsidis. Despite their similar roles in basal defense against RKN in both tested plant species, WRKY72-type transcription factors in tomato, but not in Arabidopsis, clearly contributed to basal defense against the bacterial pathogen Pseudomonas syringae. Of the five R genes that we tested in tomato and Arabidopsis, only Mi-1 appeared to be dependent on WRKY72-type transcription factors. Interestingly, AtWRKY72 target genes, identified by microarray analysis of H. arabidopsidis-triggered transcriptional changes, appear to be largely non-responsive to analogs of the defense hormone salicylic acid (SA). Thus, similarly to Mi-1, which in part acts independently of SA, AtWRKY72 appears to utilize SA-independent defense mechanisms. We propose that WRKY72-type transcription factors play a partially conserved role in basal defense in tomato and Arabidopsis, a function that has been recruited to serve Mi-1-dependent immunity.

Tomato Susceptibility to Root-Knot Nematodes Requires an Intact Jasmonic Acid Signaling Pathway

Responses of resistant (Mi-1/Mi-1) and susceptible (mi-1/ mi-1) tomato (Solanum lycopersicum) to root-knot nematodes (RKNs; Meloidogyne spp.) infection were monitored using cDNA microarrays, and the roles of salicylic acid (SA) and jasmonic acid (JA) defense signaling were evaluated in these interactions. Array analysis was used to compare transcript profiles in incompatible and compatible interactions of tomato roots 24 h after RKN infestation. The jai1 and def1 tomato mutant, altered in JA signaling, and tomato transgenic line NahG, altered in SA signaling, in the presence or absence of the RKN resistance gene Mi-1, were evaluated. The array analysis identified 1,497 and 750 genes differentially regulated in the incompatible and compatible interactions, respectively. Of the differentially regulated genes, 37% were specific to the incompatible interactions. NahG affected neither Mi-1 resistance nor basal defenses to RKNs. However, jai1 reduced tomato susceptibility to RKNs while not affecting Mi-1 resistance. In contrast, the def1 mutant did not affect RKN susceptibility. These results indicate that JA-dependent signaling does not play a role in Mi-1-mediated defense; however, an intact JA signaling pathway is required for tomato susceptibility to RKNs. In addition, low levels of SA might be sufficient for basal and Mi-1 resistance to RKNs.

The Mi-9 Gene from Solanum arcanum Conferring Heat-Stable Resistance to Root-Knot Nematodes Is a Homolog of Mi-1

Resistance conferred by the Mi-1 gene from Solanum peruvianum is effective and widely used for limiting root-knot nematode (Meloidogyne spp.) yield loss in tomato (Solanum lycopersicum), but the resistance is ineffective at soil temperatures above 28°C. Previously, we mapped the heat-stable resistance gene Mi-9 in Solanum arcanum accession LA2157 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 resistance and marker screening of an F2 population and derived F3 families from resistant LA2157 × susceptible LA392. Mi-1 intron 1 flanking primers were designed to amplify intron 1 and fingerprint Mi-1 homologs. Using these primers, we identified seven Mi-1 homologs in the mapping parents. Cf-2 and Mi-1 homologs were mapped on chromosome 6 using a subset of the F2. Cf-2 homologs did not segregate with Mi-9 resistance, but three Mi-1 homologs (RH1, RH2, and RH4) from LA2157 and one (SH1) from LA392 colocalized to the Mi-9 region. Reverse transcriptase-polymerase chain reaction analysis indicated that six Mi-1 homologs are expressed in LA2157 roots. We targeted transcripts of Mi-1 homologs for degradation with tobacco (Nicotiana tabacum) rattle virus (TRV)-based virus-induced gene silencing using Agrobacterium infiltration with a TRV-Mi construct. In most LA2157 plants infiltrated with the TRV-Mi construct, Mi-9-meditated heat-stable root-knot nematode resistance was compromised at 32°C, indicating that the heat-stable resistance is mediated by a homolog of Mi-1.

Coi1-Dependent Signaling Pathway Is Not Required for Mi-1—Mediated Potato Aphid Resistance

Tomato (Solanum lycopersicum) has a unique resistance gene, Mi-1, that confers resistance to animals from distinct taxa, nematodes, and piercing and sucking insects. Mi-1 encodes a protein with a nucleotide-binding site and leucine-rich repeat motifs. Early in the potato aphid (Macrosiphum euphorbiae)--tomato interactions, aphid feeding induces the expression of the jasmonic acid (JA)-regulated proteinase inhibitor genes, Pin1 and Pin2. The jail-1 (jasmonic acid insensitive 1) tomato mutant, which is impaired in JA perception, was used to gain additional insight into the JA signaling pathway and its role in the Mi-1-mediated aphid resistance. The jail-1 mutant has a deletion in the Coil gene that encodes a putative F-box protein. In this study, aphid colonization, survival, and fecundity were compared on wild-type tomato and jail-1 mutant. In choice assays, the jail-1 mutant showed higher colonization by potato aphids compared with wild-type tomato. In contrast, no-choice assays showed no difference in potato aphid survival or fecundity between jail-1 and the wild-type parent. Plants homozygous for Mi-1 and for the jail mutation were not compromised in resistance to potato aphids, using either choice or no-choice assays. In addition, the accumulation of JA-regulated Pin1 transcripts after aphid feeding was Coil dependent. Taken together, these data indicate that, although potato aphids activate Coil-dependent defense response in tomato, this response is not required for Mi-1-mediated resistance to aphids.

Ethylene contributes to potato aphid susceptibility in a compatible tomato host

Resistance to potato aphid (Macrosiphum euphorbiae) in tomato (Solanum lycopersicum) is conferred by Mi-1. Early during both compatible and incompatible interactions, potato aphid feeding induces the expression of ethylene (ET) biosynthetic genes. Here, we used genetic and pharmacologic approaches to investigate the role of ET signaling in basal defense and Mi-1-mediated resistance to potato aphid in tomato. The effect of potato aphid infestation on ET biosynthesis in susceptible and resistant plants was assessed. Aphid bioassays were performed using plants impaired in ET biosynthesis or perception using virus-induced gene silencing, the Never ripe (Nr) mutant, and 1-methylcyclopropene (MCP) treatment. A burst of ET was observed after aphid feeding in both resistant and susceptible plants, correlated with an increase in the expression of ET biosynthetic genes. However, impairing ET signaling or biosynthesis did not compromise Mi-1-mediated resistance but it did decrease susceptibility to potato aphid in a compatible host. 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.

Linked, if not the same, Mi-1 homologues confer resistance to tomato powdery mildew and root-knot nematodes.

On the short arm of tomato chromosome 6, a cluster of disease resistance (R) genes have evolved harboring the Mi-1 and Cf genes. The Mi-1 gene confers resistance to root-knot nematodes, aphids, and whiteflies. Previously, we mapped two genes, Ol-4 and Ol-6, for resistance to tomato powdery mildew in this cluster. The aim of this study was to investigate whether Ol-4 and Ol-6 are homologues of the R genes located in this cluster. We show that near-isogenic lines (NIL) harboring Ol-4 (NIL-Ol-4) and Ol-6 (NIL-Ol-6) are also resistant to nematodes and aphids. Genetically, the resistance to nematodes cosegregates with Ol-4 and Ol-6, which are further fine-mapped to the Mi-1 cluster. We provide evidence that the composition of Mi-1 homologues in NIL-Ol-4 and NIL-Ol-6 is different from other nematode-resistant tomato lines, Motelle and VFNT, harboring the Mi-1 gene. Furthermore, we demonstrate that the resistance to both nematodes and tomato powdery mildew in these two NIL is governed by linked (if not the same) Mi-1 homologues in the Mi-1 gene cluster. Finally, we discuss how Solanum crops exploit Mi-1 homologues to defend themselves against distinct pathogens.

Lignin Modification Leads to Increased Nodule Numbers in Alfalfa

Reducing lignin content in stems and roots of alfalfa results in an increased nodule number phenotype. Reduction of lignin levels in the forage legume alfalfa (Medicago sativa) by down-regulation of the monolignol biosynthetic enzyme hydroxycinnamoyl coenzyme A:shikimate hydroxycinnamoyl transferase (HCT) results in strongly increased digestibility and processing ability of lignocellulose. However, these modifications are often also associated with dwarfing and other changes in plant growth. Given the importance of nitrogen fixation for legume growth, we evaluated the impact of constitutively targeted lignin modification on the belowground organs (roots and nodules) of alfalfa plants. HCT down-regulated alfalfa plants exhibit a striking reduction in root growth accompanied by an unexpected increase in nodule numbers when grown in the greenhouse or in the field. This phenotype is associated with increased levels of gibberellins and certain flavonoid compounds in roots. Although HCT down-regulation reduced biomass yields in both the greenhouse and field experiments, the impact on the allocation of nitrogen to shoots or roots was minimal. It is unlikely, therefore, that the altered growth phenotype of reduced-lignin alfalfa is a direct result of changes in nodulation or nitrogen fixation efficiency. Furthermore, HCT down-regulation has no measurable effect on carbon allocation to roots in either greenhouse or 3-year field trials.

Mi-1-Mediated Resistance to Meloidogyne incognita in Tomato May Not Rely on Ethylene but Hormone Perception through ETR3 Participates in Limiting Nematode Infection in a Susceptible Host

Root-knot nematodes, Meloidogyne spp., are important pests of tomato (Solanum lycopersicum) and resistance to the three most prevalent species of this genus, including Meloidogyne incognita, is mediated by the Mi-1 gene. Mi-1 encodes a nucleotide binding (NB) leucine-rich repeat (LRR) resistance (R) protein. Ethylene (ET) is required for the resistance mediated by a subset of NB-LRR proteins and its role in Mi-1-mediated nematode resistance has not been characterized. Infection of tomato roots with M. incognita differentially induces ET biosynthetic genes in both compatible and incompatible interactions. Analyzing the expression of members of the ET biosynthetic gene families ACC synthase (ACS) and ACC oxidase (ACO), in both compatible and incompatible interactions, shows differences in amplitude and temporal expression of both ACS and ACO genes in these two interactions. Since ET can promote both resistance and susceptibility against microbial pathogens in tomato, we investigated the role of ET in Mi-1-mediated resistance to M. incognita using both genetic and pharmacological approaches. Impairing ET biosynthesis or perception using virus-induced gene silencing (VIGS), the ET-insensitive Never ripe (Nr) mutant, or 1-methylcyclopropene (MCP) treatment, did not attenuate Mi-1-mediated resistance to M. incognita. However, Nr plants compromised in ET perception showed enhanced susceptibility to M. incognita indicating a role for ETR3 in basal resistance to root-knot nematodes.

Agronomic Performance and Lignin Content of HCT Down-Regulated Alfalfa (Medicago sativa L.)

Lignin is a cell wall polymer that reduces the enzymatic digestibility and conversion efficiency of lignocellulosic biomass to ethanol. Down-regulation of the hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT) gene reduces lignin content in alfalfa (Medicago sativa L.). The objective of this study was to evaluate HCT down-regulated alfalfa plants for their forage composition and agronomic performance in the greenhouse and under field conditions. Individual plants with two separate T0HCT down-regulation events 3a and 30a were used to make crosses with a common male sterile line to generate T1 progenies (3A and 30A, respectively). The experimental design was a randomized complete block with four replications per line and ten individuals per replication. Two-row plots with full-sib progeny rows with (+) and without (−) the transgene were planted along with commercial cultivars used as checks. Most differential responses in forage quality and agronomic characteristics between full-sib down-regulated and non-down-regulated progenies were event-specific and found mainly between the HCT30A+ and HCT30A− progenies. Variation between HCT− and HCT+ alfalfa plants was observed in the HCT gene transcript levels, acid detergent lignin (ADL), relative feed value (RFV), and saccharification efficiency. Although differential responses in agronomic performance of field-grown HCT down-regulated alfalfa plants were identified for biomass yield and plant height, HCT+ and HCT− progenies had similar spring growth and fall dormancy. The reduction in lignin content of alfalfa plants via modification of HCT transcript levels increased forage quality and efficiency of sugar release of plants grown under greenhouse and field conditions.