Global gene expression profiling during Medicago truncatula-Phymatotrichopsis omnivora interaction reveals a role for jasmonic acid, ethylene, and the flavonoid pathway in disease development.

Phymatotrichopsis omnivora (Duggar) Hennebert causes a destructive root rot in cotton, alfalfa (Medicago sativa), and many other dicot species. No consistently effective control measures or resistant host germplasm for Phymatotrichum root rot (PRR) are known. The relative genetic intractability of cotton and alfalfa precludes their use as model pathosystem hosts for P. omnivora. Therefore, we used the model legume M. truncatula and its available genetic and genomic resources to investigate PRR. Confocal imaging of P. omnivora interactions with M. truncatula roots revealed that the mycelia do not form any specialized structures for penetration and mainly colonize cortical cells and, eventually, form a mycelial mantle covering the root's surfaces. Expression profiling of M. truncatula roots infected by P. omnivora identified several upregulated genes, including the pathogenesis-related class I and class IV chitinases and genes involved in reactive oxygen species generation and phytohormone (jasmonic acid and ethylene) signaling. Genes involved in flavonoid biosynthesis were induced (2.5- to 10-fold over mock-inoculated controls) at 3 days postinoculation (dpi) in response to fungal penetration. However, the expression levels of flavonoid biosynthesis genes returned to the basal levels with the progress of the disease at 5 dpi. These transcriptome results, confirmed by real-time quantitative polymerase chain reaction analyses, showed that P. omnivora apparently evades induced host defenses and may downregulate phytochemical defenses at later stages of infection to favor pathogenesis.

Genome mapping of white clover (Trifolium repens L.) and comparative analysis within the Trifolieae using cross-species SSR markers.

Allotetraploid white clover (Trifolium repens L.), a cool-season perennial legume used extensively as forage for livestock, is an important target for marker-assisted breeding. A genetic linkage map of white clover was constructed using simple sequence repeat (SSR) markers based on sequences from several Trifolieae species, including white clover, red clover (T. pratense L.), Medicago truncatula (Gaertn.) and soybean (Glycine max L.). An F(1) population consisting of 179 individuals, from a cross between two highly heterozygous genotypes, GA43 and Southern Regional Virus Resistant, was used for genetic mapping. A total of 1,571 SSR markers were screened for amplification and polymorphism using DNA from two parents and 14 F(1)s of the mapping population. The map consists of 415 loci amplified from 343 SSR primer pairs, including 83 from white clover, 181 from red clover, 77 from M. truncatula, and two from soybean. Linkage groups for all eight homoeologous chromosome pairs of allotetraploid white clover were detected. Map length was estimated at 1,877 cM with 87% genome coverage. Map density was approximately 5 cM per locus. Segregation distortion was detected in six segments of the genome (homoeologous groups A1, A2, B1, B2, C1, and D1). A comparison of map locations of markers originating from white clover, red clover, and alfalfa (M. sativa L.) revealed putative macro-colinearity between the three Trifolieae species. This map can be used to link quantitative trait loci with SSR markers, and accelerate the improvement of white clover by marker-assisted selection and breeding.

Overexpression of WXP1, a putative Medicago truncatula AP2 domain-containing transcription factor gene, increases cuticular wax accumulation and enhances drought tolerance in transgenic alfalfa (Medicago sativa).

The identification of leaf wax genes involved in stress tolerance is expected to have great potential for crop improvement. Here we report the characterization of a novel AP2 domain-containing putative transcription factor gene from the model legume Medicago truncatula. The gene, designated WXP1, is able to activate wax production and confer drought tolerance in alfalfa (Medicago sativa), the most important forage legume species in the world and a close relative of M. truncatula. The predicted protein of WXP1 has 371 aa; it is one of the longest peptides of all the single AP2 domain proteins in M. truncatula. WXP1 is distinctly different from the most studied genes in the AP2/ERF transcription factor family such as AP2s, CBF/DREB1s, DREB2s, WIN1/SHN1 and GL15. Transcript level of WXP1 is inducible by cold, abscisic acid and drought treatment mainly in shoot tissues in M. truncatula. Overexpression of WXP1 under the control of the CaMV35S promoter led to a significant increase in cuticular wax loading on leaves of transgenic alfalfa. Scanning electron microscopy revealed earlier accumulation of wax crystals on the adaxial surface of newly expanded leaves and higher densities of wax crystalline structures on both adaxial and abaxial surfaces of mature leaves. Gas chromatography-mass spectrometry analysis revealed that total leaf wax accumulation per surface area increased 29.6-37.7% in the transgenic lines, and the increase was mainly contributed by C30 primary alcohol. WXP1 overexpression induced a number of wax-related genes. Transgenic leaves showed reduced water loss and chlorophyll leaching. Transgenic alfalfa plants with increased cuticular waxes showed enhanced drought tolerance demonstrated by delayed wilting after watering was ceased and quicker and better recovery when the dehydrated plants were re-watered.