Soonok Kim

The genome sequence of the rice blast fungus Magnaporthe grisea

Magnaporthe grisea is the most destructive pathogen of rice worldwide and the principal model organism for elucidating the molecular basis of fungal disease of plants. Here, we report the draft sequence of the M. grisea genome. Analysis of the gene set provides an insight into the adaptations required by a fungus to cause disease. The genome encodes a large and diverse set of secreted proteins, including those defined by unusual carbohydrate-binding domains. This fungus also possesses an expanded family of G-protein-coupled receptors, several new virulence-associated genes and large suites of enzymes involved in secondary metabolism. Consistent with a role in fungal pathogenesis, the expression of several of these genes is upregulated during the early stages of infection-related development. The M. grisea genome has been subject to invasion and proliferation of active transposable elements, reflecting the clonal nature of this fungus imposed by widespread rice cultivation.

Vitamin B1 functions as an activator of plant disease resistance

Vitamin B1 (thiamine) is an essential nutrient for humans. Vitamin B1 deficiency causes beriberi, which disturbs the central nervous and circulatory systems. In countries in which rice (Oryza sativa) is a major food, thiamine deficiency is prevalent because polishing of rice removes most of the thiamine in the grain. We demonstrate here that thiamine, in addition to its nutritional value, induces systemic acquired resistance (SAR) in plants. Thiamine-treated rice, Arabidopsis (Arabidopsis thaliana), and vegetable crop plants showed resistance to fungal, bacterial, and viral infections. Thiamine treatment induces the transient expression of pathogenesis-related (PR) genes in rice and other plants. In addition, thiamine treatment potentiates stronger and more rapid PR gene expression and the up-regulation of protein kinase C activity. The effects of thiamine on disease resistance and defense-related gene expression mobilize systemically throughout the plant and last for more than 15 d after treatment. Treatment of Arabidopsis ecotype Columbia-0 plants with thiamine resulted in the activation of PR-1 but not PDF1.2. Furthermore, thiamine prevented bacterial infection in Arabidopsis mutants insensitive to jasmonic acid or ethylene but not in mutants impaired in the SAR transduction pathway. These results clearly demonstrate that thiamine induces SAR in plants through the salicylic acid and Ca2+-related signaling pathways. The findings provide a novel paradigm for developing alternative strategies for the control of plant diseases.

Rice defense mechanisms against Cochliobolus miyabeanus and Magnaporthe grisea are distinct

ABSTRACT Responses of rice to Magnaporthe grisea and Cochliobolus miyabeanus were compared. In Tetep, a rice cultivar resistant to both fungi, pathogen inoculation rapidly triggered the hypersensitive reaction (HR), resulting in microscopic cell death. In rice cv. Nakdong, susceptible to both pathogens, M. grisea did not cause HR, whereas C. miyabeanus caused rapid cell death similar to that associated with HR, which appeared similar to that observed in cv. Tetep, yet failed to block fungal ramification. Treatment with conidial germination fluid (CGF) from C. miyabeanus induced rapid cell death in both cultivars, suggesting the presence of phytotoxins in CGF. Pretreatment of cv. Nakdong with CGF significantly increased resistance to M. grisea, while the same treatment was ineffective against C. miyabeanus. Similarly, in cv. Nakdong, benzothiadiazole (BTH) significantly increased resistance to M. grisea, but was ineffective against C. miyabeanus. Methyl jasmonate (MeJA) treatment appeared to be ineffective against either fungus. Increased resistance of cv. Nakdong to M. grisea by BTH or CCF treatment was correlated with more rapid induction of three monitored PR genes. Application of MeJA resulted in the expression of JAmyb in cv. Nakdong being induced faster than in untreated plants in response to M. grisea infection. In contrast, the expression pattern of the PR and JAmyb genes in response to C. miyabeanus was nearly identical between cvs. Nakdong and Tetep, and neither BTH nor MeJA treatment significantly modified their expression patterns in response to C. miyabeanus infection. Our results suggest that rice employs distinct mechanisms for its defense against M. grisea versus C. miyabeanus.

Calcium/Calmodulin-Dependent Signaling for Prepenetration Development in Colletotrichum gloeosporioides

ABSTRACT Colletotrichum gloeosporioides forms a specialized infection structure, an appressorium, for host infection. Contacting hard surface induces appressorium formation in C. gloeosporioides, whereas hydrophobicity of the contact surface does not affect this infection-related differentiation. To determine if the calcium/calmodulin-dependent signaling system is involved in prepenetration morphogenesis in C. gloeosporioides pathogenic on red pepper, effects of calcium chelator (EGTA), phospholipase C inhibitor (neomycin), intracellular calcium modulators (TMB-8 and methoxy verampamil), and calmodulin antagonists (chloroproma-zine, phenoxy benzamine, and W-7) were tested on conidial germination and appressorium formation. Exogenous addition of Ca(2+), regardless of concentration, augmented conidial germination, while appressorial differentiation decreased at higher concentrations. Inhibition of appressorium formation by EGTA was partly restored by the addition of calcium ionophore A23187 or CaCl(2). Calcium channel blockers and calmodulin antagonists specifically reduced appressorium formation at micromolar levels. These results suggest that biochemical processes controlled by the calcium/calmodulin signaling system are involved in the induction of prepenetration morphogenesis in C. gloeosporioides pathogenic on red pepper.

Transformation of Phomopsis viticola with the green fluorescent protein.

Phomopsis viticola is the causal agent of Phomopsis cane and leaf spot on Vitis spp., a persistent and economically important disease in temperate regions. Here we describe the transformation of this fungus with two different constructs (pBHt2_sGFP and pIGPAPA) containing the green fluorescent protein (GFP) and the hygromycin B resistance gene (hph). Protoplast-mediated transformation yielded mitotically stable transformants with no change in virulence on grape internodes and leaves in comparison to the wild type. These transformants will be critical tools for elucidating fungal penetration of host plants, invasive growth and the nature of its host association.

Quantification of Rice Sheath Blight Progression Caused by Rhizoctonia solani

Rhizoctonia solani has a wide host range, including almost all cultivated crops and its subgroup anastomosis group (AG)-1 IA causes sheath blight in rice. An accurate measurement of pathogen’s biomass is a convincing tool for enumeration of this disease. Mycological characteristics and molecular diagnosis simultaneously supported that all six strains in this study were R. solani AG-1 IA. Heterokaryons between strains Rs40104, Rs40105, and Rs45811 were stable and viable, whereas Rs40103 and Rs40106 did not form viable fused cells, except for the combination of Rs40106 and Rs40104. A primer pair was highly specific to RsAROM gene of R. solani strains and the amplified fragment exists as double copies within fungal genome. The relationship between crossing point (CP) values and the amount of fungal DNA was reliable (R2>0.99). Based on these results, we determined R. solani’s proliferation within infected stems through real time PCR using a primer pair and a Taqman probe specific to the RsAROM gene. The amount of fungal DNA within the 250 ng of tissue DNA from rice cv. Dongjin infected with Rs40104, Rs40105, and Rs45811 were 7.436, 5.830, and 5.085 ng, respectively. In contrast, the fungal DNAs within the stems inoculated with Rs40103 and Rs40106 were 0.091 and 0.842 ng. The sheath blight symptom progression approximately coincided with the amount of fungal DNA within the symptoms. In summary, our quantitative evaluation method provided reliable and objective results reflecting the amount of fungal biomass within the infected tissues and would be useful for evaluation of resistance germplasm or fungicides and estimation of inoculum potential.

Evaluation of Fusarium head blight in barley infected by Fusarium graminearum.

Fusarium head blight, which is primarily caused by Fusarium graminearum, is a devastating disease in the barley field. A real-time PCR protocol was developed to evaluate the growth of this pathogen in the host plant tissues. All four strains harbored the gene encoding ATP-BINDING CASSETTE TRANSPORTER (FgABC; FGSG_00541) as a single copy within their genomes. Our Southern blot result was identical with the genomic data for F. graminearum strain PH-1. Based on the crossing point (CP) values obtained in our TaqMan real-time PCR analysis, two standard curves describing the relationship among the CP value, FgABC copy number, and amount of fungal DNA were constructed. Chronological enumeration of fungal growth was coincided with the symptom development.

The Application of ChIP-chip Analysis in the Rice Blast Pathogen

To attempt to gain an understanding of the molecular underpinnings of disease, many researchers have turned to expression profiling of genes during various stages of host recognition, entry, invasive growth, and host responses. While these studies have proven valuable, a deeper level of knowledge of the control circuitry affecting observed gene expression profiles can lead to a better understanding of the host pathogen interaction. Transcription factors are key switches in signal transduction circuits regulating gene expression. One powerful method to define target sequence specificity for this important group of transcription regulators is chromatin immunoprecipitation (ChIP) with microarray chips (chip), commonly called ChIP-chip. A more recent variation of this technique is ChIP-seq where DNA sequencing replaces the microarray chip. Here, we describe how we elucidated the binding sites for the Magnaporthe oryzae Ca(2+)/calcineurin-dependent transcription factor MoCRZ1 with the ChIP-chip approach.

Vegetative Compatibility Grouping and Pathogenicity of Colletotrichum gloeosporioides Isolates from Different Host Plants

A total of 57 isolates of Colletotrichum gloeosporioides were recovered from diseased tissues of Hall`s crab apple (Malus haliana), 3 cultivars of edible apple (M. pumila var. dulcissima), red pepper (Capsicum annum), and grapevine (Vitis vinifera) fruits. All isolates showed strong virulence on their own host plants. Isolates from edible apple exhibited high level of cultivar specificity in pathogenicity tests. Ten isolates from apple cultivar `Fuji` were virulent on `Jonathan` and `Rall`s Genet`. However, 12 isolates from `Jonathan` and `Rall`s Genet` were not virulent on `Fuji`. Among the 24 isolates from red pepper, only seven and two isolates were infective on edible apple and grapevine fruits, respectively. All six isolates from grapevine were only virulent on their own host. These isolates were grouped into five vegetative compatibility groups (VCGs), A, B, C, D, and E, by demonstrating heterokaryosis through complementation using nitrate-nonutilizing (nit) mutants. Among them, isolates belong to VCG-A and VCG-D accounted for 24 and 17 isolates； those in VCG-A exhibited wide host range involving Hall`s crab apple, all three edible apple cultivars, and red pepper. On the other hand, isolates of VCG-D and VCG-E showed limited host range specific to red pepper and grapevine, respectively. Taken together, the data suggest that among C. gloeosporioides isolates, the concepts of pathotype and/or forma specialis may exist, and that three is a relationship between host specificity and VCG grouping among C. gloeosporioides isolates.