Judith Lichtenzveig

The Medicago truncatula ortholog of Arabidopsis EIN2, sickle, is a negative regulator of symbiotic and pathogenic microbial associations

SUMMARY The plant hormone ethylene negatively regulates bacterial infection and nodule formation in legumes in response to symbiotic rhizobia, but the molecular mechanism(s) of ethylene action in symbiosis remain obscure. We have identified and characterized multiple mutant alleles of the MtSkl1 gene, which controls both ethylene sensitivity and nodule numbers. We show that this locus encodes the Medicago truncatula ortholog of the Arabidopsis ethylene signaling protein EIN2. In addition to the well-characterized role of MtSkl1 in rhizobial symbiosis, we show that MtSkl1 is involved in regulating early phases of the symbiotic interaction with mycorrhizal fungi, and in mediating root responses to cytokinin. MtSkl1 also functions in the defense against Rhizoctonia solani and Phytophthora medicaginis, with the latter interaction likely to involve positive feedback amplification of ethylene biosynthesis. Overexpression of the C-terminal domain of MtEIN2 is sufficient to block nodulation responses, consistent with previous reports in Arabidopsis on the activation of ethylene signaling. This same C-terminal region is uniquely conserved throughout the EIN2 homologs of angiosperms, which is consistent with its role as a higher plant-specific innovation essential to EIN2 function.

Mapping quantitative trait loci in chickpea associated with time to flowering and resistance to Didymella rabiei the causal agent of Ascochyta blight.

Drought is the major constraint to chickpea (Cicer arietinum L.) productivity worldwide. Utilizing early-flowering genotypes and advancing sowing from spring to autumn have been suggested as strategies for drought avoidance. However, Ascochyta blight (causal agent: Didymella rabiei (Kov.) v. Arx.) is a major limitation for chickpea winter cultivation. Most efforts to introgress resistance to the pathogen into Kabuli germplasm resulted in relatively late flowering germplasm. With the aim to explore the feasibility of combining earliness and resistance, RILs derived from a cross between a Kabuli cultivar and a Desi accession were evaluated under field conditions and genotyped with SSR markers. Three quantitative trait loci (QTLs) with significant effects on resistance were identified: two linked loci located on LG4 in epistatic interaction and a third locus on LG8. Two QTLs were detected for time to flowering: one in LG1 and another on LG2. When resistance and time to flowering were analyzed together, the significance of the resistance estimates obtained for the LG8 locus increased and the locus effect on days to flowering, previously undetected, was significantly different from zero. The identification of a locus linked both to resistance and time to flowering may account for the correlation observed between these traits in this and other breeding attempts.

The B-3 ethylene response factor MtERF1-1 mediates resistance to a subset of root pathogens in Medicago truncatula without adversely affecting symbiosis with rhizobia.

The fungal necrotrophic pathogen Rhizoctonia solani is a significant constraint to a range of crops as diverse as cereals, canola, and legumes. Despite wide-ranging germplasm screens in many of these crops, no strong genetic resistance has been identified, suggesting that alternative strategies to improve resistance are required. In this study, we characterize moderate resistance to R. solani anastomosis group 8 identified in Medicago truncatula. The activity of the ethylene- and jasmonate-responsive GCC box promoter element was associated with moderate resistance, as was the induction of the B-3 subgroup of ethylene response transcription factors (ERFs). Genes of the B-1 subgroup showed no significant response to R. solani infection. Overexpression of a B-3 ERF, MtERF1-1, in Medicago roots increased resistance to R. solani as well as an oomycete root pathogen, Phytophthora medicaginis, but not root knot nematode. These results indicate that targeting specific regulators of ethylene defense may enhance resistance to an important subset of root pathogens. We also demonstrate that overexpression of MtERF1-1 enhances disease resistance without apparent impact on nodulation in the A17 background, while overexpression in sickle reduced the hypernodulation phenotype. This suggests that under normal regulation of nodulation, enhanced resistance to root diseases can be uncoupled from symbiotic plant-microbe interactions in the same tissue and that ethylene/ERF regulation of nodule number is distinct from the defenses regulated by B-3 ERFs. Furthermore, unlike the stunted phenotype previously described for Arabidopsis (Arabidopsis thaliana) ubiquitously overexpressing B-3 ERFs, overexpression of MtERF1-1 in M. truncatula roots did not show adverse effects on plant development.

Two alternative recessive quantitative trait loci influence resistance to spring black stem and leaf spot in Medicago truncatula

BackgroundKnowledge of the genetic basis of plant resistance to necrotrophic pathogens is incomplete and has been characterised in relatively few pathosystems. In this study, the cytology and genetics of resistance to spring black stem and leaf spot caused by Phoma medicaginis, an economically important necrotrophic pathogen of Medicago spp., was examined in the model legume M. truncatula.ResultsMacroscopically, the resistant response of accession SA27063 was characterised by small, hypersensitive-like spots following inoculation while the susceptible interaction with accessions A17 and SA3054 showed necrotic lesions and spreading chlorosis. No unique cytological differences were observed during early infection (<48 h) between the resistant and susceptible genotypes, except pathogen growth was restricted to one or a few host cells in SA27063. In both interactions reactive oxygen intermediates and phenolic compounds were produced, and cell death occurred. Two F2 populations segregating for resistance to spring black stem and leaf spot were established between SA27063 and the two susceptible accessions, A17 and SA3054. The cross between SA27063 and A17 represented a wider cross than between SA27063 and SA3054, as evidenced by higher genetic polymorphism, reduced fertility and aberrant phenotypes of F2 progeny. In the SA27063 × A17 F2 population a highly significant quantitative trait locus (QTL, LOD = 7.37; P < 0.00001) named resistance to the necrotroph Phomamedicaginis one (rnpm1) genetically mapped to the top arm of linkage group 4 (LG4). rnpm1 explained 33.6% of the phenotypic variance in the populations response to infection depicted on a 1–5 scale and was tightly linked to marker AW256637. A second highly significant QTL (LOD = 6.77; P < 0.00001), rnpm2, was located on the lower arm of LG8 in the SA27063 × SA3054 map. rnpm2 explained 29.6% of the phenotypic variance and was fine mapped to a 0.8 cM interval between markers h2_16a6a and h2_21h11d. rnpm1 is tightly linked to a cluster of Toll/Interleukin1 receptor-nucleotide binding site-leucine-rich repeat (TIR-NBS-LRR) genes and disease resistance protein-like genes, while no resistance gene analogues (RGAs) are apparent in the genomic sequence of the reference accession A17 at the rnpm2 locus.ConclusionThe induction of defence responses and cell death in the susceptible interaction following infection by P. medicaginis suggested this pathogen is not negatively affected by these responses and may promote them. A QTL for resistance was revealed in each of two populations derived from crosses between a resistant accession and two different susceptible accessions. Both loci are recessive in nature, and the simplest explanation for the existence of two separate QTLs is the occurrence of host genotype-specific susceptibility loci that may interact with undetermined P. medicaginis virulence factors.

Genetic diversity and population structure of Ascochyta rabiei from the western Iranian Ilam and Kermanshah provinces using MAT and SSR markers

Knowledge of genetic diversity in A. rabiei provides different levels of information that are important in the management of crop germplasm resources. Gene flow on a regional level indicates a significant potential risk for the regional spread of novel alleles that might contribute to fungicide resistance or the breakdown of resistance genes. Simple sequence repeat (SSR) and mating type (MAT) markers were used to determine the genetic structure, and estimate genetic diversity and the prevalence of mating types in 103 Ascochyta rabiei isolates from seven counties in the Ilam and Kermanshah provinces of western Iran (Ilam, Aseman abad, Holaylan, Chardavol, Dareh shahr, Gilangharb, and Sarpul). A set of 3 microsatellite primer pairs revealed a total of 75 alleles; the number of alleles varied from 15 to 34 for each marker. A high level of genetic variability was observed among A. rabiei isolates in the region. Genetic diversity was high (He = 0.788) within populations with corresponding high average gene flow and low genetic distances between populations. The smallest genetic distance was observed between isolates from Ilam and Chardavol. Both mating types were present in all populations, with the majority of the isolates belonging to Mat1-1 (64%), but within populations the proportions of each mating type were not significantly different from 50%. Results from this study will be useful in breeding for Ascochyta blight-resistant cultivars and developing necessary control measures.

Distribution of mating types and diversity in virulence of Didymella rabiei in Israel.

The distribution of mating types and diversity in virulence of Didymella rabiei populations were studied in Israel from 1997 to 1999. Forty-one monoconidial D. rabiei isolates from 18 commercial fields distributed among all the chickpea production areas of the country were paired with MAT1-1 and MAT1-2 mating type tester isolates of D. rabiei. Both mating types were found in all chickpea production areas of the country. Of the 18 fields sampled, MAT1-1 was observed in 44%, and MAT1-2 in 88% of the sites. In some sites both mating types were present in close proximity, suggesting that sexual reproduction of the pathogen was feasible. The contribution of sexual reproduction of the fungus to virulence diversity was tested on detached leaves of six differential chickpea cultivars. Nine isolates were derived from different well separated foci (derived from ascospores as inoculum) and eight isolates were derived from a single, well defined infection focus (derived from sister conidia). In the analyses of variance the cultivar × isolate interaction showed no significant (P of F>0.09) effect on disease incidence; the chickpea cultivars differed significantly (P of F<0.0001) in their response to D. rabiei; and the isolate effect was highly significant (P of F = 0.0007) for the conidial population, but not significant (P of F>0.1) among isolates of the ascosporic population. Nevertheless, when comparing a cultivar at a time, the ascosporic and conidial populations did not differ significantly (P of F>0.1) in their virulence diversity. Virulence of 41 isolates collected from the different chickpea fields was tested on detached leaves of four Israeli cultivars that differ in their field response to D. rabiei. The cultivar × isolate interaction showed no significant effect (P of F = 0.95) on disease incidence. The main effects of cultivar and isolate on disease incidence were highly significant (P of F<0.0001). Accordingly, our data do not support the hypothesis that there is pathogenic specialization in the D. rabiei–C. arietinum pathosystem in Israel.

Towards the First Linkage Map of the Didymella rabiei Genome

A genetic map was developed for the ascomyceteDidymella rabiei (Kovachevski) v. Arx (anamorph:Ascochyta rabiei Pass. Labr.), the causal agent of Ascochyta blight in chickpea (Cicer arietinum L.). The map was generated with 77 F1 progeny derived from crossing an isolate from the U.S.A. and an isolate from Syria. A total of 232 DAF (DNA Amplification Fingerprinting) primers and 37 STMS (Sequence-Tagged Microsatellite Site) primer pairs were tested for polymorphism between the parental isolates; 50 markers were mapped, 36 DAFs and 14 STMSs. These markers cover 261.4cM in ten linkage groups. Nineteen markers remained unlinked. Significant deviation from the expected 1:1 segregation ratios was observed for only two markers (Prob. of χ2<0.05). The implications of our results on ploidy level of the asexual spores are discussed.

Associations between earliness, Ascochyta response, and grain yield in chickpea

Evidence from an array of dryland systems suggests that chickpea (Cicer arietinum L.) grain yield could be improved through better phenological adaptation. However, information on the relationship between phenology and Ascochyta response genes, and their possible interaction with biomass and grain yield, is missing. Accordingly, the aim of the present study was to determine the associations between the above factors and biomass and grain yield in chickpea. To that end, standard Israeli cultivars and advanced generation bulked progeny from the cv. Hadas × ICC5810 cross were used. Hadas is a late-flowering, high-yielding Israeli kabuli (0.45 g/seed) cultivar with moderate field resistance to Ascochyta blight, whereas ICC5810 is a day-neutral desi (0.15 g/seed) genotype with a strong temperature response, from India. Higher yields were observed among the late-flowering bulks of the Hadas × ICC5810 progeny. No relationship between the Ascochyta response and biomass and grain yield was observed. No interaction between the phenology and Ascochyta response grouping on biomass and grain yield was observed. The results demonstrate the feasibility of combining Ascochyta resistance with earlier flowering and its potential to improve chickpea adaptation to dryland systems.