Plant-pathogen interactions: leaf physiology alterations in poplars infected with rust (Melampsora medusae).

Rust produced by Melampsora sp. is considered one of the most relevant diseases in poplar plantations. Growth reduction in poplar plantations takes place because rust, like other pathogens, alters leaf physiology. There is not a complete evaluation of several of the physiological traits that can be affected by rust at leaf level. Therefore, the aim of this work was to evaluate, in an integrative way and in the same pathosystem, which physiological processes are affected when Populus deltoides Bartr. ex Marsh. leaves are infected by rust (Melampsora medusae Thümen). Leaves of two clones with different susceptibility to rust were analyzed. Field and pot experiments were performed, and several physiological traits were measured in healthy and infected leaves. We conclude that rust affects leaf mesophyll integrity, and so water movement in the leaf in liquid phase is affected. As a consequence, gas exchange is reduced, affecting both carbon fixation and transpiration. However, there is an increase in respiration rate, probably due to plant and fungal respiration. The increase in respiration rate is important in the reduction of net photosynthetic rate, but also some damage in the photosynthetic apparatus limits leaf capacity to fix carbon. The decrease in chlorophyll content would start later and seems not to explain the reduction in net photosynthetic rate. Both clones, although they have different susceptibility to rust, are affected in the same physiological mechanisms.

Genetic transformation and expression of transgenic lines of Populus x euramericana with insect-resistance and salt-tolerance genes.

We characterized new transgenic varieties of poplar with multiple insect-resistant and salt stress tolerant genes. Two insect-resistant Bacillus thuringiensis (Bt) genes, Cry1Ac and Cry3A, and a salt-tolerant gene, Betaine aldehyde dehydrogenase (BADH) were inserted into a vector, p209-Cry1Ac-Cry3A-BADH. The clone of Populus x euramericana was transformed by the vector using the Agrobacterium-mediated method. Three transgenic lines were assessed using genetic detection and resistance expression analysis. PCR revealed that exogenous genes Cry1Ac, Cry3A, BADH and selective marker gene NPTII were present in three transgenic lines. Quantitative real-time PCR (qPCR) showed significant differences in the transcriptional abundance of three exogenous genes in different lines. Results of assays for Bt toxic proteins showed that the Cry1Ac and Cry3A toxic protein content of each line was 12.83-26.32 and 2108.91-2724.79 ng/g, respectively. The Cry1Ac toxic protein content of different lines was significantly different; the Cry3A toxic protein content was about 100 times higher than that of the Cry1Ac toxic protein. The insect-resistance test revealed the mortality rate of transgenic lines to Hyphantria cunea L1 larvae varied by 42.2-66.7%, which was significantly higher than non-transgenic lines. The mortality rate of L1 and L2 Plagiodera versicolora larvae was 100%. The insecticidal effect of transgenic lines to P. versicolora larvae was higher than that to H. cunea larvae. NaCl stress tolerance of three transgenic lines under 3-6% NaCl concentration was significantly higher than that of non-transgenic lines.

RNA silencing in the model mycorrhizal fungus Laccaria bicolor: gene knock-down of nitrate reductase results in inhibition of symbiosis with Populus.

Mycorrhizal symbioses are a rule in nature and may have been crucial in plant and fungal evolution. Ectomycorrhizas are mutualistic interactions between tree roots and soil fungi typical of temperate and boreal forests. The functional analysis of genes involved in developmental and metabolic processes, such as N nutrition, is important to understand the ontogeny of this mutualistic symbiosis. RNA silencing was accomplished in the model mycorrhizal fungus Laccaria bicolor by Agrobacterium-mediated gene transfer. Promoter-directed expression of double-stranded RNA with a partial coding sequence of the Laccaria nitrate reductase gene resulted in fungal transgenic strains strongly affected in growth with nitrate as N source in a medium with high concentration of an utilizable C source. The phenotype correlated with a clear reduction of the target gene mRNA level and this effect was not caused by homologous recombination of the T-DNA in the nitrate reductase locus. Transformation with the hairpin sequence resulted in specific CpG methylation of both the silenced transgene and the nitrate reductase encoding gene. The methylation in the target gene was restricted to the silencing trigger sequence and did not represent the entire genomic DNA in the dikaryon suggesting that the epigenetic changes accompanying RNA silencing affected only the transformed nucleus. Mycorrhization experiments of Populus with strongly silenced fungal strains revealed a systematic inhibition of symbiosis under mycorrhization conditions (C starvation) and nitrate as N source compared with the wild type. This inhibition of mycorrhization was reversed by an organic N source only utilizable by the fungus. These observations would indicate that the plant may be capable of monitoring and detecting the nutritional status of a potential symbiont avoiding the establishment of an unsatisfactory interaction. A probable control mechanism conducted by the plant would inhibit symbiosis when the metabolic profile of the fungal partner is not proper and mutual benefit from the symbiotic structure cannot be assured. Our results are the first report showing that the alteration of expression of a fungal gene impairs mycorrhization. Moreover, this work is the first demonstration of RNA silencing in mycorrhizal fungi and clearly shows that gene knock-down is a powerful tool for further functional genomic studies in mycorrhizal research.