Sang-Ryeol Park

Overexpression of the mitogen-activated protein kinase gene OsMAPK33 enhances sensitivity to salt stress in rice ( Oryza sativa L.)

Mitogen-activated protein kinases (MAPK) signalling cascades are activated by extracellular stimuli such as environmental stresses and pathogens in higher eukaryotic plants. To know more about MAPK signalling in plants, a MAPK cDNA clone, OsMAPK33, was isolated from rice. The gene is mainly induced by drought stress. In phylogenetic analysis, OsMAPK33 (Os02g0148100) showed approximately 47–93% identity at the amino acid level with other plant MAPKs. It was found to exhibit organ-specific expression with relatively higher expression in leaves as compared with roots or stems, and to exist as a single copy in the rice genome. To investigate the biological functions of OsMAPK33 in rice MAPK signalling, transgenic rice plants that either overexpressed or suppressed OsMAPK33 were made. Under dehydration conditions, the suppressed lines showed lower osmotic potential compared with that of wild-type plants, suggesting a role of OsMAPK33 in osmotic homeostasis. Nonetheless, the suppressed lines did not display any significant difference in drought tolerance compared with their wild-type plants. With increased salinity, there was still no difference in salt tolerance between OsMAPK33-suppressed lines and their wild-type plants. However, the overexpressing lines showed greater reduction in biomass accumulation and higher sodium uptake into cells, resulting in a lower K+/Na+ ratio inside the cell than that in the wild-type plants and OsMAPK33-suppressed lines. These results suggest that OsMAPK33 could play a negative role in salt tolerance through unfavourable ion homeostasis. Gene expression profiling of OsMAPK33 transgenic lines through rice DNA chip analysis showed that OsMAPK33 altered expression of genes involved in ion transport. Further characterization of downstream components will elucidate various biological functions of this novel rice MAPK.

Genetic engineering of drought-resistant tobacco plants by introducing the trehalose phosphorylase (TP) gene from Pleurotus sajor-caju

This study generated transgenic tobacco plants expressing trehalose phosphorylase of Pleurotus sajor-caju (PsTP) constitutively under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Sixteen transgenic lines were selected by genomic Southern blot analysis for further study. Unlike yeast TPS1-transformed or Escherichia coli TPS1-transformed tobacco or potato, all of the PsTP transgenic tobacco lines showed normal growth phenotypes both in the culture tubes and soil mixture. The study measured the trehalose contents of PsTP-transformed tobacco plants as well as the wild type and empty vector-transformed control plants. Results showed that the PsTP transformant contained 6.3 μmol g−1 of plant tissues, while the wild type and the control plants had only minimal levels of trehalose. Because this study detected a significant amount of trehalose in PsTP transgenic tobacco plants, it decided to carry out a bioanalysis of the PsTP transgenic tobacco plants by drought treatment by not watering the plants for over 10 days. A significant difference in drought resistance was observed from the second nonwatering day between the transgenic and the control tobacco plants. The transgenic tobacco plants had normal growth and did not wither, while the wild type and the only empty vector-transformed control plants withered severely. Among all the transgenic lines, line 10-4 showed the strongest resistance to drought stress. It did not wither even after 10 days without watering. In addition, when the drought resistance of PsTP transgenic tobacco plants was tested using detached leaves, most transgenic plants, except one line, showed better capacity to retain water than the empty vector-transformed transgenic plant.

Understanding the plant-pathogen interactions in the context of proteomics-generated apoplastic proteins inventory

The extracellular space between cell wall and plasma membrane acts as the first battle field between plants and pathogens. Bacteria, fungi, and oomycetes that colonize the living plant tissues are encased in this narrow region in the initial step of infection. Therefore, the apoplastic region is believed to be an interface which mediates the first crosstalk between host and pathogen. The secreted proteins and other metabolites, derived from both host and pathogen, interact in this apoplastic region and govern the final relationship between them. Hence, investigation of protein secretion and apoplastic interaction could provide a better understanding of plant-microbe interaction. Here, we are briefly discussing the methods available for the isolation and normalization of the apoplastic proteins, as well as the current state of secretome studies focused on the in-planta interaction between the host and the pathogen.

Transcriptome Analysis of Early Responsive Genes in Rice during Magnaporthe oryzae Infection

Rice blast disease caused by Magnaporthe oryzae is one of the most serious diseases of cultivated rice (Oryza sativa L.) in most rice-growing regions of the world. In order to investigate early response genes in rice, we utilized the transcriptome analysis approach using a 300 K tilling microarray to rice leaves infected with compatible and incompatible M. oryzae strains. Prior to the microarray experiment, total RNA was validated by measuring the differential expression of rice defense-related marker genes (chitinase 2, barwin, PBZ1, and PR-10) by RT-PCR, and phytoalexins (sakuranetin and momilactone A) with HPLC. Microarray analysis revealed that 231 genes were up-regulated (>2 fold change, p < 0.05) in the incompatible interaction compared to the compatible one. Highly expressed genes were functionally characterized into metabolic processes and oxidation-reduction categories. The oxidative stress response was induced in both early and later infection stages. Biotic stress overview from MapMan analysis revealed that the phytohormone ethylene as well as signaling molecules jasmonic acid and salicylic acid is important for defense gene regulation. WRKY and Myb transcription factors were also involved in signal transduction processes. Additionally, receptor-like kinases were more likely associated with the defense response, and their expression patterns were validated by RT-PCR. Our results suggest that candidate genes, including receptor-like protein kinases, may play a key role in disease resistance against M. oryzae attack.

Priming by Rhizobacterium Protects Tomato Plants from Biotrophic and Necrotrophic Pathogen Infections through Multiple Defense Mechanisms

A selected strain of rhizobacterium, Pseudomonas putida strain LSW17S (LSW17S), protects tomato plants (Lycopersicon esculentum L. cv. Seokwang) from bacterial speck by biotrophic Pseudomonas syringae pv. tomato strain DC3000 (DC3000) and bacterial wilt by necrotrophic Ralstonia solanacearum KACC 10703 (Rs10703). To investigate defense mechanisms induced by LSW17S in tomato plants, transcription patterns of pathogenesis-related (PR) genes and H2O2 production were analyzed in plants treated with LSW17S and subsequent pathogen inoculation. LSW17S alone did not induce transcriptions of employed PR genes in leaves and roots. DC3000 challenge following LSW17S triggered rapid transcriptions of PR genes and H2O2 production in leaves and roots. Catalase infiltration with DC3000 attenuated defense-related responses and resistance against DC3000 infection. Despite depriving H2O2 production and PR1b transcription by the same treatment, resistance against Rs10703 infection was not deterred significantly. H2O2 is indispensable for defense signaling and/or mechanisms primed by LSW17S and inhibition of bacterial speck, however, it is not involved in resistance against bacterial wilt.

OsWRKY51, a rice transcription factor, functions as a positive regulator in defense response against Xanthomonas oryzae pv. oryzae

Key messageOsWRKY51 functions as a positive transcriptional regulator in defense signaling againstXanthomonas oryzae pv. oryzaeby direct DNA binding to the promoter of defense related gene, OsPR10a.AbstractOsWRKY51 in rice (Oryza sativa L.) is induced by exogenous salicylic acid (SA) and inoculation with Xanthomonas oryzae pv. oryzae (Xoo). To examine the role of OsWRKY51 in the defense response of rice, we generated OsWRKY51 overexpressing and underexpressing transgenic rice plants. OsWRKY51-overexpressing transgenic rice lines were more resistant to Xoo and showed greater expression of defense-related genes than wild-type (WT) plants, while OsWRKY51-underexpressing lines were more susceptible to Xoo and showed less expression of defense-associated genes than WT plants. Transgenic lines overexpressing OsWRKY51 showed growth retardation compared to WT plants. In contrast, transgenic lines underexpressing OsWRKY51 by RNA interference showed similar plant height with WT plants. Transient expression of OsWRKY51-green fluorescent protein fusion protein in rice protoplasts revealed that OsWRKY51 was localized in the nucleus. OsWRKY51 bound to the W-box and WLE1 elements of the OsPR10a promoter. Based on these results, we suggest that OsWRKY51 is a positive transcriptional regulator of defense signaling and has direct DNA binding ability to the promoter of OsPR10a, although it is reported to be a negative regulator in GA signaling.

Arabidopsis Cell Death in Compatible and Incompatible Interactions with Alternaria brassicicola

Two strains of necrotrophic Alternaria brassicicola, Ab40857 and Ab42464, are virulent on Korean cabbage and several wild types of Arabidopsis thaliana. Interaction between Ab42464 and Col-0 was compatible, whereas interaction between Ab40857 and Col-0 was incompatible. The loss of defense, no death (dnd) 1 function abrogated the compatibility between Ab42464 and Col-0, and the accelerated cell death (acd) 2 mutation attenuated the Col-0’s resistance against Ab40857. These two fungal strains induced PR1 transcription in Col-0. Ab40857 accelerated transcription of PDF1.2, THI2.1, CAT, and POX by 12 h compared to those challenged with Ab42464. More abundant cell death was observed in Col-0 infected with Ab42464, however, callose deposition was evident in the incompatible interaction. Remarkably, Ab40857-infected areas of acd2-2 underwent rampant cell death and Ab42464 triggered callose production in dnd1-1. Furthermore, the incompatibility between Ab40857 and Col-0 was nullified by the coronatine-insensitive 1 (coi1) and phytoalexin-deficient 3 (pad3) mutations but not by nonexpresser of PR genes (npr1) and pad4. Ab40857 induced abundant cell death in pad3. Taken together, cell death during the early infection stage is a key determinant that discriminates between a compatible interaction and an incompatible one, and the resistance within Col-0 against Ab40857 is dependent on a defense-signaling pathway mediated by jasmonic acid and PAD3.

Overexpression of a Pathogenesis-Related Protein 10 Enhances Biotic and Abiotic Stress Tolerance in Rice.

Pathogenesis-related proteins play multiple roles in plant development and biotic and abiotic stress tolerance. Here, we characterize a rice defense related gene named “jasmonic acid inducible pathogenesis-related class 10” (JIOsPR10) to gain an insight into its functional properties. Semi-quantitative RT-PCR analysis showed up-regulation of JIOsPR10 under salt and drought stress conditions. Constitutive over-expression JIOsPR10 in rice promoted shoot and root development in transgenic plants, however, their productivity was unaltered. Further experiments exhibited that the transgenic plants showed reduced susceptibility to rice blast fungus, and enhanced salt and drought stress tolerance as compared to the wild type. A comparative proteomic profiling of wild type and transgenic plants showed that overexpression of JIOsPR10 led to the differential modulation of several proteins mainly related with oxidative stresses, carbohydrate metabolism, and plant defense. Taken together, our findings suggest that JIOsPR10 plays important roles in biotic and abiotic stresses tolerance probably by activation of stress related proteins.

Secreted Alpha-N-Arabinofuranosidase B Protein Is Required for the Full Virulence of Magnaporthe oryzae and Triggers Host Defences

Rice blast disease caused by Magnaporthe oryzae is one of the most devastating fungal diseases of rice and results in a huge loss of rice productivity worldwide. During the infection process, M. oryzae secretes a large number of glycosyl hydrolase proteins into the host apoplast to digest the cell wall and facilitate fungal ingression into host tissues. In this study, we identified a novel arabinofuranosidase-B (MoAbfB) protein that is secreted by M. oryzae during fungal infection. Deletion of MoAbfB from M. oryzae resulted in reduced disease severity in rice. Biochemical assays revealed that the MoAbfB protein exhibited arabinofuranosidase activity and caused degradation of rice cell wall components. Interestingly, pre-treatment of rice with the MoAbfB protein inhibited fungal infection by priming defence gene expression. Our findings suggest that MoAbfB secretion affects M. oryzae pathogenicity by breaking down the host cell wall, releasing oligosaccharides that may be recognized by the host to trigger innate immune responses.

Quantification of Rice Brown Leaf Spot through Taqman Real-Time PCR Specific to the Unigene Encoding Cochliobolus miyabeanus SCYTALONE DEHYDRATASE1 Involved in Fungal Melanin Biosynthesis

Rice brown leaf spot is a major disease in the rice paddy field. The causal agent Cochliobolus miyabeanus is an ascomycete fungus and a representative necrotrophic pathogen in the investigation of rice-microbe interactions. The aims of this research were to identify a quantitative evaluation method to determine the amount of C. miyabeanus proliferation in planta and determine the method’s sensitivity. Real-time polymerase chain reaction (PCR) was employed in combination with the primer pair and Taqman probe specific to CmSCD1, a C. miyabeanus unigene encoding SCYTALONE DEHYDRATASE, which is involved in fungal melanin biosynthesis. Comparative analysis of the nucleotide sequences of CmSCD1 from Korean strains with those from the Japanese and Taiwanese strains revealed some sequence differences. Based on the crossing point (CP) values from Taqman real-time PCR containing a series of increasing concentrations of cloned amplicon or fungal genomic DNA, linear regressions with a high level of reliability (R2>0.997) were constructed. This system was able to estimate fungal genomic DNA at the picogram level. The reliability of this equation was further confirmed using DNA samples from both resistant and susceptible cultivars infected with C. miyabeanus. In summary, our quantitative system is a powerful alternative in brown leaf spot forecasting and in the consistent evaluation of disease progression.