IR64, the most widely grown indica rice in South and Southeast Asia, possesses many positive agronomic characteristics (e.g., wide adaptability, high yield potential, tolerance to multiple diseases and pests, and good eating quality,) that make it an ideal genotype for identifying mutational changes in traits of agronomic importance. We have produced a large collection of chemical and irradiation-induced IR64 mutants with different genetic lesions that are amenable to both forward and reverse genetics. About 60,000 IR64 mutants have been generated by mutagenesis using chemicals (diepoxybutane and ethylmethanesulfonate) and irradiation (fast neutron and gamma ray). More than 38,000 independent lines have been advanced to M4 generation enabling evaluation of quantitative traits by replicated trials. Morphological variations at vegetative and reproductive stages, including plant architecture, growth habit, pigmentation and various physiological characters, are commonly observed in the four mutagenized populations. Conditional mutants such as gain or loss of resistance to blast, bacterial blight, and tungro disease have been identified at frequencies ranging from 0.01% to 0.1%. Results from pilot experiments indicate that the mutant collections are suitable for reverse genetics through PCR-detection of deletions and TILLING. Furthermore, deletions can be detected using oligomer chips suggesting a general technique to pinpoint deletions when genome-wide oligomer chips are broadly available. M4 mutant seeds are available for users for screening of altered response to multiple stresses. So far, more than 15,000 mutant lines have been distributed. To facilitate broad usage of the mutants, a mutant database has been constructed in the International Rice Information System (IRIS; http: //www.iris.irri.org) to document the phenotypes and gene function discovered by users.

SummaryIn lower eukaryotes, beta-oxidation of fatty acids is restricted primarily to the peroxisomes and the resultant acetyl-CoA molecules (and the chain-shortened fatty acids) are transported via the cytosol into the mitochondria for further breakdown and usage. Using a loss-of-function mutation in the Magnaporthe grisea PEROXIN6 orthologue, we define an essential role for peroxisomal acetyl-CoA during the host invasion step of the rice-blast disease. We show that an Mgpex6 D strain lacks functional peroxisomes and is incapable of b -oxidation of long-chain fatty acids. The Mgpex6 D mutant lacked appressorial melanin and host penetration, and was completely non-pathogenic. We further show that a peroxisome-associated carnitine acetyl-transferase (Crat1) activity is essential for such appressorial function in Magnaporthe . CRAT1 -minus appressoria showed reduced melanization, but were surprisingly incapable of elaborating penetration pegs or infection hyphae. Exogenous addition of excess glucose during infection stage caused partial remediation of the pathogenicity defects in the crat1 D strain. Moreover, Mgpex6 D and crat1 D mycelia showed increased sensitivity to Calcofluor white, suggesting that weakened cell wall biosynthesis in a glucose-deficient environment leads to appressorial dysfunction in these mutants. Interestingly, CRAT1 was itself essential for growth on acetate and longchain fatty acids. Thus, carnitine-dependent metabolic activities associated with the peroxisomes, cooperatively facilitate the appressorial function of host invasion during rice-blast infections.

The Woronin body is a peroxisome-derived dense-core vesicle that is specific to several genera of filamentous ascomycetes, where it has been shown to seal septal pores in response to cellular damage. The Hexagonal peroxisome (Hex1) protein was recently identified as a major constituent of the Woronin body and shown to be responsible for selfassembly of the dense core of this organelle. Using a mutation in the Magnaporthe grisea HEX1 ortholog, we define a dual and essential function for Woronin bodies during the pathogenic phase of the rice blast fungus. We show that the Woronin body is initially required for proper development and function of appressoria (infection structures) and subsequently necessary for survival of infectious fungal hyphae during invasive growth and host colonization. Fungal mycelia lacking HEX1 function were unable to survive nitrogen starvation in vitro, suggesting that in planta growth defects are a consequence of the mutant's inability to cope with nutritional stress. Thus, Woronin body function provides the blast fungus with an important defense against the antagonistic and nutrient-limiting environment encountered within the host plant.