Seong-Sook Han

A Putative MAP Kinase Kinase Kinase, MCK1, Is Required for Cell Wall Integrity and Pathogenicity of the Rice Blast Fungus, Magnaporthe oryzae

Insertional mutagenesis of Magnaporthe oryzae led to the identification of MCK1, a pathogenicity gene predicted to encode mitogen-activated protein kinase kinase kinase (MAPKKK) homologous to BCK1 in Saccharomyces cerevisiae. Targeted disruption of MCK1 resulted in the fungus undergoing autolysis and showing hypersensitivity to cell-wall-degrading enzyme. The mck1 produced significantly reduced numbers of conidia and developed appressoria in a slightly retarded manner compared with the wild type. Appressorium of the mck1 mutant was unable to penetrate into plant tissues, thereby rendering the mutant nonpathogenic. Cytorrhysis assay and monitoring of lipid mobilization suggested that the appressorial wall was altered, presumably affecting the level of turgor pressure within appressorium. Furthermore, the mck1 mutant failed to grow inside plant tissue. Complementation of the mutated gene restored its ability to cause disease symptoms, demonstrating that MCK1 is required for fungal pathogenicity. Taken together, our results suggest that MCK1 is an MAPKKK involved in maintaining cell wall integrity of M. oryzae, and that remodeling of the cell wall in response to host environments is essential for fungal pathogenesis.

Genome‐wide analysis of T‐DNA integration into the chromosomes of Magnaporthe oryzae

A grobacterium tumefaciens‐ mediated transformation (ATMT) has become a prevalent tool for functional genomics of fungi, but our understanding of T‐DNA integration into the fungal genome remains limited relative to that in plants. Using a model plant‐pathogenic fungus, Magnaporthe oryzae, here we report the most comprehensive analysis of T‐DNA integration events in fungi and the development of an informatics infrastructure, termed a T‐DNA analysis platform (TAP). We identified a total of 1110 T‐DNA‐tagged locations (TTLs) and processed the resulting data via TAP. Analysis of the TTLs showed that T‐DNA integration was biased among chromosomes and preferred the promoter region of genes. In addition, irregular patterns of T‐DNA integration, such as chromosomal rearrangement and readthrough of plasmid vectors, were also observed, showing that T‐DNA integration patterns into the fungal genome are as diverse as those of their plant counterparts. However, overall the observed junction structures between T‐DNA borders and flanking genomic DNA sequences revealed that T‐DNA integration into the fungal genome was more canonical than those observed in plants. Our results support the potential of ATMT as a tool for functional genomics of fungi and show that the TAP is an effective informatics platform for handling data from large‐scale insertional mutagenesis.

Distribution of Xanthomonas oryzae pv. oryzae Strains Virulent to Xa21 in Korea

ABSTRACT Strains of Xanthomonas oryzae pv. oryzae that are virulent to rice lines carrying the Xa21 resistance gene were widely distributed in Korea. A total of 105 strains collected during 1987 to 1996 in Korea was characterized by pathogenicity tests and restriction fragment length polymorphism analysis of the XorII methyltransferase (xorIIM) and avrXa10 genes. Although the lesion lengths on rice line IRBB21, which carries Xa21, decreased as plant age increased, resistance and susceptibility of the plants to 31 strains were clearly differentiated at the seedling (14, 21, and 28 days old), maximum tillering, and flag leaf stages. The resistance or susceptibility of seedlings was correlated with bacterial populations within an inoculated leaf. There was a significant change in the population structure of X. oryzae pv. oryzae with regard to virulence to Xa21 over the last 10 years; this change in population was confirmed by genome analysis. Lineage I, which is avirulent to Xa21 and does not have a genomic xorIIM homolog, was the predominant lineage found between 1987 and 1989, while lineage II, which is virulent to Xa21 and contains the xorIIM homolog, was predominant in strains collected between 1994 and 1995. Our results demonstrate that introduction of Xa21 into commercial rice should be based on the regional structure of X. oryzae pv. oryzae populations and suggest that Xa21 will not be useful in Korea.

The Pi40 Gene for Durable Resistance to Rice Blast and Molecular Analysis of Pi40-Advanced Backcross Breeding Lines

Rice blast severely reduces production in both irrigated and water-stressed upland ecosystems of tropical and temperate countries. Nearly 50 blast resistance genes have been identified and some of those are incorporated into several rice cultivars. However, most of the resistance genes break down in a few years because of their race specificity and the rapid change in pathogenicity of the blast fungus (Magnaporthe grisea). The objective of this study was to analyze advanced backcross breeding lines (ABL) possessing the gene Pi40 for durable rice blast resistance. In all, 4 resistant genotypes, 4 japonica cultivars, and 10 monogenic differential rice genotypes with some known resistance genes were bioassayed in the greenhouse using seven sequential plantings and 29 virulent M. grisea isolates of Korea. The genotypes with the Pi40 gene had <3% diseased leaf area, which was significantly below the disease threshold level of 40% considered for durable blast resistance. Moreover, the genotypes with the Pi40 gene expressed compatibility with only two to three virulent M. grisea isolates supporting durability of resistance, in contrast to susceptible cultivars with >50% diseased leaf area and 10 compatible isolates. Of the 10 known resistance genes tested, Piz-t, Piz-5, and Pi9 showed differential reactions to the pathogen isolates in seven plantings. Genotyping of the ABL with 260 simple sequence repeat (SSR) markers revealed rapid conversion toward recurrent parent genotypes with fewer donor chromosomal segments (5.3 to 14.5%). Our study based on a sequential testing and background selection of breeding lines with the resistance gene Pi40 provided valuable information for durable blast resistance breeding in rice.

Diversity of Pathotypes and DNA Fingerprint Haplotypes in Populations of Magnaporthe grisea in Korea over Two Decades.

ABSTRACT Using isolates collected over 2 decades, we determined the population structure and dynamics of the rice blast fungus, Magnaporthe grisea, in Korea at both the genotypic and phenotypic levels. Pathotype analysis on 6,315 isolates collected from 328 rice cultivars from 1981 to 2000 revealed the presence of a total of 91 pathotypes. Among these 91 patho-types, nine dominated, comprising 76.5% of the isolates. The expected number of pathotypes (corrected for sample size) increased significantly during the course of this study. On average, six (ranging from 0 to 20) new commercial cultivars were introduced annually between 1981 and 1998. However, the overall cultivar diversity, estimated using the Shannon index, was low. Most of the new cultivars were not planted to a large area because the seven most common cultivars each year occupied over 70% of the rice-cultivated area. The frequencies of the nine dominant patho-types from these seven cultivars were highly correlated with those from the entire set of cultivars. To understand genetic diversity within and between pathotypes, 176 isolates collected from 1984 to 1999 were randomly sampled and analyzed by DNA fingerprinting. High similarities were observed among isolates; overall similarities were greater than 63% in combined MGR586 and MAGGY DNA fingerprints. Unlike most other populations of M. grisea, DNA fingerprints showed no clear lineage structure. No groups were supported by bootstrap values greater than 10%. Furthermore, there was no significant correlation between DNA fingerprint similarities and pathotypes. Genetic similarity was significantly greater (P < 0.001) within years than between years, although the difference was small. Our data suggest that M. grisea populations in Korea have been mostly dominated by a single clonal lineage. We cannot conclude from these data that selection by the host population has been a major force in the evolution of M. grisea in Korea.

Potent in Vivo Antifungal Activity against Powdery Mildews of Pregnane Glycosides from the Roots of Cynanchum wilfordii

Two new pregnane glycosides, kidjoranine 3-O-β-D-glucopyranosyl-(1 → 4)-β-D-glucopyranosyl-(1 → 4)-α-L-cymaropyranosyl-(1 → 4)-β-D-cymaropyranosyl-(1→4)-α-L-diginopyranosyl-(1 → 4)-β-D-cymaropyranoside (5) and caudatin 3-O-β-D-glucopyranosyl-(1 → 4)-β-D-glucopyranosyl-(1 → 4)-α-L-cymaropyranosyl-(1 → 4)-β-D-cymaropyranosyl-(1 → 4)-α-L-diginopyranosyl-(1 → 4)-β-D-cymaropyranoside (6), were isolated from the roots of Cynanchum wilfordii along with four known compounds (1-4). The antifungal activities of the six compounds against barley powdery mildew caused by Blumeria graminis f. sp. hordei were compared to the antifungal activity of polyoxin B. The caudatin glycosides (1, 4, and 6) showed stronger antifungal activities than polyoxin B, whereas kidjoranine glycosides (2, 3, and 5) had weaker activities than polyoxin B. A wettable powder-type formulation (C. wilfordii-WP20) of the ethyl acetate extract from C. wilfordii roots prohibited the development of barley powdery mildew much more effectively than the commercial fungicide polyoxin B-WP10. In addition, C. wilfordii-WP20 effectively controlled strawberry powdery mildew caused by Sphaerotheca humuli under greenhouse conditions. Thus, the crude extract containing the pregnane glycosides can be used as a botanical fungicide for the environmentally benign control of powdery mildews.

Comparative Analysis of Pathogenicity and Phylogenetic Relationship in Magnaporthe grisea Species Complex

Outbreaks of rice blast have been a threat to the global production of rice. Members of the Magnaporthe grisea species complex cause blast disease on a wide range of gramineous hosts, including cultivated rice and other grass species. Recently, based on phylogenetic analyses and mating tests, isolates from crabgrass were separated from the species complex and named M. grisea. Then other isolates from grasses including rice were named as M. oryzae. Here, we collected 103 isolates from 11 different species of grasses in Korea and analyzed their phylogenetic relationships and pathogenicity. Phylogenetic analyses of multilocus sequences and DNA fingerprinting revealed that the haplotypes of most isolates were associated with their hosts. However, six isolates had different haplotypes from the expectation, suggesting potential host shift in nature. Results of pathogenicity tests demonstrated that 42 isolates from crabgrass and 19 isolates from rice and other grasses showed cross-infectivity on rice and crabgrass, respectively. Interestingly, we also found that the isolates from rice had a distinct deletion in the calmodulin that can be used as a probe.

Genetic differentiation of Magnaporthe oryzae populations from scouting plots and commercial rice fields in Korea.

A previous study of the diversity and population structure of the rice blast fungus, Magnaporthe oryzae, over a 20-year period in Korea, found novel fingerprint haplotypes each year, and the authors hypothesized that populations might experience annual bottlenecks. Based on this model, we predicted that M. oryzae populations would have little or no genetic differentiation among geographic regions because rice blast is commonly found throughout Korea each year and M. oryzae would have to disperse from small populations surviving annually between rice crops. To test this hypothesis, we sampled M. oryzae from rice fields in eight provinces in Korea in a single year (1999). In four provinces, we sampled from a set of rice cultivars commonly grown in commercial fields (group I); because of low disease incidence in four other provinces, we could not sample from commercial fields and instead sampled from scouting plots of different cultivars set up for detecting new pathotypes of M. oryzae (group II). All isolates were genotyped with DNA fingerprint probes MGR586 and MAGGY, a telomere-linked gene family member TLH1, the PWL2 host specificity gene and mating type. Fingerprint haplotypes clustered into two distinct lineages corresponding to the two sets of cultivars (groups I and II), with haplotype similarities of 71% between lineages and >76% within lineages. Isolates from the same cultivar within group I were genetically differentiated among locations, and isolates within the same location were differentiated among cultivars. Differentiation for TLH1 and PWL2 was significant (P < 0.03), but not as strong as for fingerprint markers. Similar analyses were not possible among group II isolates because too few isolates were available from any one cultivar. All isolates were in the same mating type, Mat1-1, ruling out sexual reproduction as a source of novel haplotypes. When the 1999 samples were compared with the historical samples from the previous study, haplotypes of group I formed a separate cluster, while those of group II clustered with haplotypes from the historical sample. Altogether, geographic subdivision, monomorphism of mating type, and correlation of haplotypes to sets of cultivars are not consistent with the hypothesis of repeated turnover of haplotypes. Instead, the previous correlations of haplotypes to year might have been caused by inadequate sampling of haplotypes each year, highlighting the need for studies of population genetics to be conducted with systematic samples collected to address specific questions.

Damage Analysis of Rice Panicle Blast on Disease Occurrence Time and Severity

The structural differences between healthy and diseasedpanicle necks caused by Pyricularia oryzae were observed using electron-microscope. In the diseased panicle neck, the infection hyphae of the rice blast pathogen grew through the sclerenchymatous fiber tissue and reached to the central internal lacuna. Since the pathogen grew through the sclerenchymatous fiber tissues, the vascular bundle composed with xylem and phloem had been destroyed and finally the nutrients from the leaf and stem were not able to be transported into the grains. Infection of panicle base by the blast pathogen until 20 days after heading caused more than 50% of yield loss in both Jinmibyeo and Chucheongbyeo. There was a positive correlation between incidence of the panicle blast and rice yield losses. The regression equations between incidence of the panicle blast and yield losses were y

Genetic Stability of Magnaporthe oryzae during Successive Passages through Rice Plants and on Artificial Medium

Department of Plant Pathology, The Pennsylvania State University, University Park 16802, USA(Received on September 27, 2010; Accepted on November 2, 2010)Genetic instability of the rice blast fungus Magnaportheoryzae has been suggested as a major factor underlyingthe rapid breakdown of host resistance in the field.However, little information is available on the mech-anism of genetic instability. In this study, we assessedthe stability of repetitive DNA elements and several keyphenotypic traits important for pathogenesis afterserially transferring two isolates though rice plants andan artificial medium. Using isolate 70-15, we obtained atotal of 176 single-spore isolates from 10 successiverounds of culturing on artificial medium. Another 20isolates were obtained from germ tubes formed at thebasal and apical cells of 10 three-celled conidia. Addi-tionally, 60 isolates were obtained from isolate KJ201after serial transfers through rice plants and an arti-ficial medium. No apparent differences in phenotypes,including mycelial growth, conidial morphologies, coni-diation, conidial germination, appressorium formation,and virulence, or in DNA fingerprints using MGR586,MAGGY, Pot2, LINE, MG-SINE and PWL2 as probeswere observed among isolates from the same parent iso-late. Southern hybridization and sequence analysis oftwo avirulence genes, AVR-Pita1 and AVR-Pikm, show-ed that both genes were also maintained stably during10 successive generations on medium and plants. How-ever, one reversible loss of restriction fragments wasfound in the telomere-linked helicase gene ( TLH1) family,suggesting some telomere regions may be more unstablethan the rest of the genome. Taken together, our resultssuggest that phenotype and genotype of M. oryzae iso-lates do not noticeably change, at least up to 10 successivegenerations on a cultural medium and in host plants.Keywords :genetic stability, genotypes, Magnaporthe grisea,phenotypes, repeat sequencesMagnaporthe oryzae is a heterothallic ascomycete fungusthat causes rice blast, one of the most devastating diseasesin rice throughout the world. Since there is no evidence forsexual reproduction in the field in most parts of the world,asexual spores (conidia) are the most important source ofinoculum. Through the production of conidia, M. oryzaenormally undergoes 8-11 disease cycles in rice plants underfield conditions during one growing season (Han et al.,1997). Although asexual reproduction is expected to pro-duce genetically identical clones, the high rate of appear-ance of new races with the ability to infect previouslyresistant varieties of rice, suggests that M. oryzae may havehigh mutation rates in avirulence genes, which determinerace (also called pathotype in M. oryzae) (Kang et al., 2001;Kang and Lee, 2000; Zhou et al., 2007).Although the most effective control strategy for rice blastis cultivating resistant varieties, it has often achieved onlyshort-term success due to frequent breakdown of newlyintroduced resistance. This resistance breakdown has beenattributed to genetic variability in M. oryzae (Bonman et al.,1986; Correa-Victoria and Zeigler, 1993; Correa-Victoriaand Zeigler, 1993). Various mechanisms have been sug-gested to explain the frequent generation of race variants,including heterokaryosis, parasexuality, and mutations(Genovesi and Magill, 1976; Wu and Magill, 1995; Xia andCorrell, 1995; Zeigler et al., 1997). Parasexual recombina-tion of genetic markers, resulting new genetic variants, hasbeen demonstrated in the laboratory for M. oryzae, sug-gesting the potential for parasexual recombination togenerate new races (Noguchi et al., 2006; Zeigler et al.,1997). However, no evidence for parasexuality has beenfound in nature for M. oryzae. The activity of transposableDNA elements is another possible mechanism that has beensuggested (Kang et al., 2001; Nishimura et al., 2000; Zhouet al., 2007). For example, mobilization of Pot2, Pot3, andLINE elements has been shown to cause variation by insert-ing into promoter region or coding sequences of avirulence