María I. Oloriz

Sugarcane genes differentially expressed in response to Puccinia melanocephala infection: identification and transcript profiling

Brown rust caused by the fungus Puccinia melanocephala is a major disease of sugarcane (Saccharum spp.). A sugarcane mutant, obtained by chemical mutagenesis of the susceptible variety B4362, showed a post-haustorial hypersensitive response (HR)-mediated resistance to the pathogen and was used to identify genes differentially expressed in response to P. melanocephala via suppression subtractive hybridization (SSH). Tester cDNA was derived from the brown rust-resistant mutant after inoculation with P. melanocephala, while driver cDNAs were obtained from the non-inoculated resistant mutant and the inoculated susceptible donor variety B4362. Database comparisons of the sequences of the SSH recombinant clones revealed that, of a subset of 89 non-redundant sequences, 88% had similarity to known functional genes, while 12% were of unknown function. Thirteen genes were selected for transcript profiling in the resistant mutant and the susceptible donor variety. Genes involved in glycolysis and C4 carbon fixation were up-regulated in both interactions probably due to disturbance of sugarcane carbon metabolism by the pathogen. Genes related with the nascent polypeptide associated complex, post-translational proteome modulation and autophagy were transcribed at higher levels in the compatible interaction. Up-regulation of a putative l-isoaspartyl O-methyltransferase S-adenosylmethionine gene in the compatible interaction may point to fungal manipulation of the cytoplasmatic methionine cycle. Genes coding for a putative no apical meristem protein, S-adenosylmethionine decarboxylase, non-specific lipid transfer protein, and GDP-l-galactose phosphorylase involved in ascorbic acid biosynthesis were up-regulated in the incompatible interaction at the onset of haustorium formation, and may contribute to the HR-mediated defense response in the rust-resistant mutant.

Selection and characterisation of sugarcane mutants with improved resistance to brown rust obtained by induced mutation

The brown rust susceptible sugarcane genotype B4362 was subjected to in vitro tissue culture and physical and chemical mutation induction procedures. Five brown rust resistant mutants with hypersensitive response to Puccinia melanocephala were selected out of a total population of 11 167 regenerated plants. High selection frequency was obtained with both mutagenic treatments, although chemical mutagenesis (NaN3) resulted in higher selection frequencies for brown rust resistance than gamma irradiation (60Co). The brown rust resistant mutants showed variations in molecular, morphological, and agronomic traits. Traits such as internode shape, bud shape, leaf sheath hairiness, outer auricule shape, intensity of flowering, stool growth habit, number of stalks per stool, and smut susceptibility were modified in brown rust resistant mutants. In addition, sugar yield was improved in two mutants with increments in stalk length, stalk number, and stalk diameter. Mutation induction proved to be suitable for the generation of new sources of brown rust resistance in sugarcane.

Early regulation of primary metabolism, antioxidant, methyl cycle and phenylpropanoid pathways during the Mycosphaerella fijiensis-Musa spp. interaction

Black leaf streak disease (BLSD) caused by Mycosphaerella fijiensis is considered the most destructive and costly foliar disease that affects bananas and plantains. In spite of some recent progress regarding the study of M. fijiensis-Musa spp. interaction, there is still limited information for this pathosystem. To gain insights into the molecular mechanisms behind the M. fijiensis-Musa spp. interaction, gene expression and biochemical analysis related with primary metabolism, methyl cycle, antioxidant and phenylpropanoid pathways were conducted. Early screening of the resistant cultivar (cv.) ‘Calcutta 4’ (Musa AA) and the susceptible cv. ‘Grande naine’ (Musa AAA) plants infected with M. fijiensis was effective at looking for defense-related genes. Quantitative PCR experiments showed up-regulation of Photosystem I reaction center subunit N chloroplastic-like (primary metabolism) and S-adenosyl-L-methionine synthetaste (methyl cycle) genes in the incompatible interaction, as well as down-regulation of the phenylpropanoid pathway genes in the susceptible cv. ‘Grande naine’ as main finding of this study. Improved knowledge concerning the M. fijiensis-Musa spp. interaction could help to establish innovative approaches for plant breeding programs against BLSD.