Expression of the wheat multipathogen resistance hexose transporter Lr67res is associated with anion fluxes

OnlinePublMany disease resistance genes in wheat (Triticum aestivum L.) confer strong resistance to specific pathogen races or strains, and only a small number of genes confer multipathogen resistance. The Leaf rust resistance 67 (Lr67) gene fits into the latter category as it confers partial resistance to multiple biotrophic fungal pathogens in wheat and encodes a Sugar Transport Protein 13 (STP13) family hexose-proton symporter variant. Two mutations (G144R, V387L) in the resistant variant, Lr67res, differentiate it from the susceptible Lr67sus variant. The molecular function of the Lr67res protein is not understood, and this study aimed to broaden our knowledge on this topic. Biophysical analysis of the wheat Lr67sus and Lr67res protein variants was performed using Xenopus laevis oocytes as a heterologous expression system. Oocytes injected with Lr67sus displayed properties typically associated with proton-coupled sugar transport proteins—glucose-dependent inward currents, a Km of 110 ± 10 µM glucose, and a substrate selectivity permitting the transport of pentoses and hexoses. By contrast, Lr67res induced much larger sugar-independent inward currents in oocytes, implicating an alternative function. Since Lr67res is a mutated hexose-proton symporter, the possibility of protons underlying these currents was investigated but rejected. Instead, currents in Lr67res oocytes appeared to be dominated by anions. This conclusion was supported by electrophysiology and 36Cl− uptake studies and the similarities with oocytes expressing the known chloride channel from Torpedo marmorata, TmClC-0. This study provides insights into the function of an important disease resistance gene in wheat, which can be used to determine how this gene variant underpins disease resistance in planta.Ricky J. Milne, Katherine E. Dibley, Jayakumar Bose, Anthony R. Ashton, Peter R. Ryan, Stephen D. Tyerman, and Evans S. Laguda

Variability of phenylalanine ammonia-lyase and peroxidase activities in leaves of subterranean clover is determined by their susceptibility to Kabatiella caulivora

Kabatiella caulivora is the causal organism of northern anthracnose or clover (Trifolium spp.) scorch disease. The activities of phenylalanine ammonia-lyase (PAL) and soluble peroxidase were determined in seedling leaves of two cultivars of subterranean clover (T.subterraneum) inoculated with race 1 or race 2 of K. caulivora. A small increase in activity of PAL was recorded in both cultivars 2–4 h post inoculation with either race. A second,large increase in PAL activity was observed only in the incompatible interaction (cv. Daliak inoculated with race 1), increasing 18-fold between 8 and 48 h post inoculation. Peroxidase activity in cv. Daliak increased rapidly within 2 h post inoculation with either race but was significantly higher in the incompatible interaction. Peroxidase activity in cv. Woogenellup increased by 4 h post inoculation with either race, but was significantly lower than that in cv. Daliak. Subsequent increases in peroxidase activity were recorded in both cultivars, however the levels remained constant in cv. Daliak infected with race 1, while activities in the other race-cultivar combinations decreased to control levels. It is hypothesised that the peak of activity of PAL at 48 h, and the rapid increase in peroxidase at 2 h are related to the race-specific resistance response of cv. Daliak to race 1 of K. caulivora, and that minor peaks of activity in the compatible interactions are general defence responses

Infection of Subterranean Clover (Trifolium subterraneum) by Kabatiella caulivora

Kabatiella caulivora is a serious pathogen of clover (Trifolium) spp. Subterranean clover (T. subterraneum) cv. Woogenellup was inoculated with K. caulivora, to study the attachment and germination of conidia, germ-tube penetration of the plant surface, and histochemistry and ultrastructure of changes in the host associated with lesion development. The foliar architecture caused the conidia to concentrate at the base of leaflets and on the petiolules (between the leaflets and petioles). Epidermal cells immediately beneath conidia and, occasionally, also adjacent cells developed a yellow-brown discoloration 1 day post-inoculation. Penetration appeared to be directly through the cuticle, characterized by constricted hyphae at the point of entry. No appressoria were observed. In leaves, invasion was restricted to the area proximal to the petiolule and leaf mid-rib. In petioles and petiolules, the hyphae initially remained between the epidermal cells and first layer of mesophyll cells before moving intercellularly through the mesophyll tissue towards phloem tissues. The cuticle was occasionally degraded in petiole and petiolule infections, the loss of epidermal and mesophyll cell wall components was detected, and chloroplasts and starch grains were disrupted. Plants developed macroscopic symptoms 10-11 days post-inoculation with necrotic lesions occurring on leaves, petioles and petiolules. Sporulation occurred approximately 15-18 days post-inoculation when affected plants collapsed. This information may be useful for breeding programmes aimed at selecting varieties with improved resistance to the clover scorch disease

Variability within Kabatiella caulivora Race 1 and Race 2 revealed by cultural and molecular analyses

Kabatiella caulivora is the causal agent of clover scorch, a fungal disease of clover (Trifolium) species. Variability within and between K. caulivora Race 1 and Race 2 was determined by cultural characteristics, isozymes, and amplified fragment length polymorphisms (AFLP). Cultural studies indicated isolates from both races were highly variable. No differences were identified within or between races by isozyme analysis. Similarity coefficients, determined from AFLP analysis, indicated that isolates from different races were often more similar than isolates from the same race. Comparison of single representative isolates from Race 1 and Race 2, collected at a Denmark (Western Australia) disease site, with isolates collected from another site of clover scorch outbreak at Esperance, 300 km east of Denmark, indicated most of the isolates causing the second outbreak were similar to Race 2, confirming previously conducted pathogenicity tests. It is hypothesised that Race 2 may have evolved from Race 1, and that the level of variability in the pathogen indicates the potential for development of further new races of K. caulivora. The requirement for improved selection strategies, including the screening of new cultivars and breeding lines with multiple isolates of the pathogen, is discussed in relation to these findings

Differences in symptom development in subterranean clover infected with Kabatiella caulivora Race 1 and Race 2 are related to host resistance

Clover scorch (Kabatiella caulivora) is a severe fungal disease of Trifolium spp. contributing to the collapse of pasture swards across southern Australia during warm, humid spring weather. Host plant responses associated with resistance to the disease were determined in 2 cultivars of subterranean clover (T. subterraneum) separately inoculated with K. caulivora Race 1 or Race 2. Germination of conidia of both races reached a maximum 5 days post-inoculation on cv. Woogenellup (susceptible to both races) and 4 days post-inoculation on cv. Daliak (resistant to Race 1 but susceptible to Race 2). Germ tube growth of Race 1 was inhibited on cv. Daliak and the percentage of conidia penetrating leaf surfaces was lowest on this race–cultivar combination. Susceptibility was characterised by large petiole lesions, with invasive hyphae extending through the mesophyll tissue into the pith and then through the phloem tissue of vascular bundles, eventually causing the petioles to collapse. Resistance was characterised by small, black lesions with invasive hyphae extending no further than the fourth layer of mesophyll cells. A suberin-based material was observed beneath infected mesophyll cells in the incompatible interaction, beyond which no further growth of hyphae occurred. Race 2 caused a faster rate of host tissue necrosis than Race 1 and also the breakdown of starch grains in uninvaded petiole tissues. Starch grains in plants infected with Race 1 were evenly distributed in uninvaded tissue. Sporulation was rarely observed in the incompatible interaction but was common in compatible interactions within 15 days post-inoculation. These responses to K. caulivora can now be used as a breeding tool in evaluating and selecting improved resistance to clover scorch disease among breeding lines of subterranean clover

Genetic variability in a collection of Stagonospora nodorum isolates from Western Australia

Stagonospora nodorum isolates were collected from the Western Australian grain-belt during 1993. These isolates and a subset of isolates taken from a single location were used to assay the level of variation within the pathogen population. The isolates were compared using anonymous nuclear DNA markers. Three low copy-number and a single high copy-number RFLP probe were used to generate polymorphisms. The collection exhibited a high genotypic diversity for the high copy-number probe, a result consistent with the high level of sexual reproduction previously found in the fungal population. The high level of genotypic diversity was consistent with previous international studies. There was no evidence of differentiation between the total collection of isolates and the subset of isolates taken from the single location. Further work needs to be undertaken to determine if the aggressiveness of the pathogen is influenced by the host genotype

A single gene controls resistance to septoria nodorum blotch in the Aegilops tauschii accession AUS21712

A potential source of resistance to septoria nodorum blotch had been identified in an accession of the wild wheat, Aegilops tauschii. A cross was made between the resistant Ae. tauschii accession, AUS21712, and a susceptible accession, CPI110889, to study the genetics of resistance. The parental accessions and the F1, F3, and F4 progeny were screened in the glasshouse as seedlings. The resistant parent took significantly longer to develop symptoms, developed significantly fewer lesions, and expressed significantly lower levels of disease than the susceptible parent. The F1 mean response for disease severity indicated resistance was dominant. The genotypic ratios generated from the screening of the F3 and F4 generations were not significantly different from the genotypic ratio expected for a single gene. The efficacy of the resistance and its simple genetic control in the Ae. tauschii accession AUS21712 means that the potential exists to use this Ae. tauschii resistance gene in a bread wheat breeding program

Identification of a molecular marker linked to Septoria nodorum blotch resistance in Triticum tauschii using F2 bulked segregant

A search was conducted for a molecular marker linked to a gene for resistance to septoria nodorum blotch in the Triticum tauschii accession RL5271. DNA was extracted from leaves of F2 plants which were progeny tested to identify homozygous resistant and homozygous susceptible F2 plants. The DNA from the homozygous resistant plants was pooled together and the low copy sequences were enriched using renaturation kinetics and hydroxyapatite to remove the repetitive DNA sequences. The pooled DNA from the homozygous susceptible plants was treated in the same manner. The pooled DNA and the parental DNA were screened using RAPD primers. Two markers present in the pooled resistant DNA and in the parental DNA were identified and cloned. These markers were verified using RFLPs with the cloned marker as a probe. One of the probes did not produce any polymorphism as a RFLP, even when a number of different restriction enzymes were used. The second marker was polymorphic between the two parents when the DNA was restricted with HindIII and then MseI. In the 14 homozygous F2 plants tested, the marker was completely linked to the resistance gene. This marker may be valuable in introgressing the resistance gene from T. tauschii into a commercial bread wheat cultivar