Yuhko Kobayashi

Differential Activation of the Rice Sucrose Nonfermenting1–Related Protein Kinase2 Family by Hyperosmotic Stress and Abscisic Acid

To date, a large number of sequences of protein kinases that belong to the sucrose nonfermenting1–related protein kinase2 (SnRK2) family are found in databases. However, only limited numbers of the family members have been characterized and implicated in abscisic acid (ABA) and hyperosmotic stress signaling. We identified 10 SnRK2 protein kinases encoded by the rice (Oryza sativa) genome. Each of the 10 members was expressed in cultured cell protoplasts, and its regulation was analyzed. Here, we demonstrate that all family members are activated by hyperosmotic stress and that three of them are also activated by ABA. Surprisingly, there were no members that were activated only by ABA. The activation was found to be regulated via phosphorylation. In addition to the functional distinction with respect to ABA regulation, dependence of activation on the hyperosmotic strength was different among the members. We show that the relatively diverged C-terminal domain is mainly responsible for this functional distinction, although the kinase domain also contributes to these differences. The results indicated that the SnRK2 protein kinase family has evolved specifically for hyperosmotic stress signaling and that individual members have acquired distinct regulatory properties, including ABA responsiveness by modifying the C-terminal domain.

Loss-of-Function Mutations of the Rice GAMYB Gene Impair α-Amylase Expression in Aleurone and Flower Development

GAMYB was first isolated as a positive transcriptional regulator of gibberellin (GA)-dependent α-amylase expression in barley aleurone cells, and its molecular and biochemical properties have been well characterized. However, the role of GAMYB elsewhere in the plant is not well understood. To investigate the molecular function of GAMYB outside of the aleurone cells, we isolated loss-of-function mutants from a panel of rice mutants produced by the insertion of a retrotransposon, Tos17. Through PCR screening using primers for rice GAMYB (OsGAMYB) and Tos17, we isolated three independent mutant alleles that contained Tos17 inserted in the exon region. No α-amylase expression in the endosperm was induced in these mutants in response to GA treatment, indicating that the Tos17 insertion had knocked out OsGAMYB function. We found no significant defects in the growth and development of the mutants at the vegetative stage. After the phase transition to the reproductive stage, however, shortened internodes and defects in floral organ development, especially a defect in pollen development, were observed. On the other hand, no difference was detected in flowering time. High-level OsGAMYB expression was detected in the aleurone cells, inflorescence shoot apical region, stamen primordia, and tapetum cells of the anther, but only low-level expression occurred in organs at the vegetative stage or in the elongating stem. These results demonstrate that, in addition to its role in the induction of α-amylase in aleurone, OsGAMYB also is important for floral organ development and essential for pollen development.

α-Tubulin is Rapidly Phosphorylated in Response to Hyperosmotic Stress in Rice and Arabidopsis

By using high-resolution two-dimensional PAGE followed by phosphoprotein-specific staining and peptide mass fingerprint analysis along with other assays, we found that α-tubulin is phosphorylated in response to hyperosmotic stress in rice and Arabidopsis. The onset of the phosphorylation response was as early as 2 min after hyperosmotic stress treatment, and a major proportion of α-tubulin was phosphorylated after 60 min in root tissues. However, the phosphorylated form of α-tubulin was readily dephosphorylated upon stress removal. The phosphorylation site was identified as Thr349 by comprehensive mutagenesis of serine/threonine residues in a rice α-tubulin isoform followed by evaluation in cultured cell protoplasts. This residue is located at the surface for the interaction with β-tubulin in polymerized α-β tubulin dimers and has been proposed to be directly involved in this interaction. Thus, α-tubulin phosphorylation was considered to occur on free tubulin dimers in response to hyperosmotic stress. The incorporation of green fluorescent protein (GFP)-α-tubulin into cortical microtubules was completely inhibited in transgenic Arabidopsis when Thr349 was substituted with glutamate or aspartate. Using transgenic Arabidopsis plants expressing GFP-α-tubulin, we found that hyperosmotic stress causes extensive cortical microtubule depolymerization. Microtubule-destabilizing treatments such as propyzamide or oryzalin and temperature stresses resulted in α-tubulin phosphorylation, whereas hyperosmotic stress-induced α-tubulin phosphorylation was partially inhibited by taxol, which stabilizes microtubules. These results and the three-dimensional location of the phosphorylation site suggested that microtubules are depolymerized in response to hyperosmotic stress via α-tubulin phosphorylation. Together, the results of the present study reveal a novel mechanism that globally regulates the microtubule polymerization.

Depolymerization of the actin cytoskeleton induces defense responses in tobacco plants

Tobacco leaf sections were treated with actin inhibitors, i.e., cytochalasins, to determine the effects of actin depolymerization on tobacco defense responses. Inoculation of the leaf sections with the pathogen Erysiphe cichoracearum, depolymerized the actin cytoskeleton, priming the cells for a hypersensitive response-like cell death. Further, expression of the acidic PR1 and PR2 genes were induced in cytochalasin-treated leaf sections. The intensity of the cytochalasin effects on the defense responses was closely correlated with the extent of actin depolymerization. This suggests that plant cells may perceive perturbation of the actin cytoskeleton, and this stimulus may trigger plant defense responses.

Microtubules and Pathogen Defence

The cytoskeletal network of plant cells represents a dynamic structure that responds to external stimuli by changes of organization. An attack of pathogenic microbes represents an external stress that seriously threatens plant survival. Growing evidence from recent research indicates that cytoskeletal elements, such as microtubules and microfilaments, are central players in plant defence responses. Tubulin and actin inhibitors suppress the polarization of cellular events related to plant defence, such as massive cytoplasmic aggregation, deposition of papillae and the accumulation of autofluorescent compounds at the sites of fungal penetration. Simultaneously, these inhibitors allow non-pathogenic fungi to penetrate successfully into non-host plants. Thus, microtubules and microfilaments, through the temporal and spatial regulation of molecules and/or organelles in the host cell, seem to control responses conferring resistance to attempted fungal penetration. In addition, elements of the plant cytoskeleton seem to play a critical role in hypersensitive cell death. On the other hand, several plant pathogens produce anti-cytoskeletal compounds during invasion, suggesting that the plant cytoskeleton represents an advantageous target for plant pathogens and symbionts. The possibility of enhancing plant resistance to pathogens via artificial manipulation of cytoskeletal elements will be discussed.

Calcium ion promotes successful penetration of powdery mildew fungi into barley cells

To determine whether Ca2+ promotes powdery mildew penetration, Ca2+-treated barley coleoptiles were inoculated with conidia of pathogenic and nonpathogenic fungi. Penetration efficiency of the pathogenic powdery mildew Blumeria graminis was enhanced by Ca2+ treatment, but that of the necrotrophic pathogen Helminthosporium sp. remained unaffected. Similarly, when actin-dependent penetration resistance is suppressed with cytochalasin A, Ca2+ treatment specifically enhanced penetration of the nonpathogenic powdery mildew Erysiphe pisi but not that of other nonpathogens. Calmodulin inhibitors suppressed the promotive effect of Ca2+ on B. graminis penetration. These results suggest that barley powdery mildew specifically requires Ca2+ and calmodulin for penetration.

Increased Expression of the Tomato SISWEET15 Gene During Grey MoldInfection and the Possible Involvement of the Sugar Efflux to Apoplasm inthe Disease Susceptibility

Host plant susceptibility genes, which facilitate pathogen growth during plant infection, are attractive targets for disease-resistance breeding. To explore candidate susceptibility genes in tomatoes during Botrytis cinerea infection, the fungal infection-responsive SWEET genes were screened for out of all 31 tomato SlSWEET genes. The expression of only one gene, SlSWEET15, was induced by B. cinerea at the pre-necrotic stage (16 h post inoculation), whereas most of the other SWEET genes were downregulated. The expression of the SlSWEET15 transiently increased by 16 h post inoculation, then reduced to basal levels by 24 h post inoculation. We measured the glucose and sucrose contents of fluid of infected cotyledons at the pre-necrotic stage (20 h post inoculation). The sugar contents of the apoplasmic fluids were significantly higher in the infected cotyledons compared to 0 h. Furthermore, glucose and sucrose can promote growth and invasion of B. cinerea both in vitro and in vivo. SWEET proteins in clade III, including the deduced SlSWEET15, are well-known sugar efflux transporters. These results suggest that SlSWEET15 is induced by B. cinerea and that this is exploited by the fungus, which may provide sugars to promote hyphal growth in the pre-necrotic stage of infection in tomato.

Successful identification of a predictive biomarker for lymph node metastasis in colorectal cancer using a proteomic approach

Colorectal cancer (CRC)-associated mortality is primarily caused by lymph node (LN) and distant metastasis, highlighting the need for biomarkers that predict LN metastasis and facilitate better therapeutic strategies. We used an Isobaric Tags for Relative and Absolute Quantification (iTRAQ)-based comparative proteomics approach to identify novel biomarkers for predicting LN metastasis in CRC patients. We analyzed five paired samples of CRC with or without LN metastasis, adjacent normal mucosa, and normal colon mucosa, and differentially expressed proteins were identified and subsequently validated at the protein and/or mRNA levels by immunohistochemistry and qRT-PCR, respectively. We identified 55 proteins specifically associated with LN metastasis, from which we selected ezrin for further analysis and functional assessment. Expression of ezrin at both the protein and mRNA levels was significantly higher in CRC tissues than in adjacent normal colonic mucosa. In univariate analysis, high ezrin expression was significantly associated with tumor progression and poor prognosis, which was consistent with our in vitro findings that ezrin promotes the metastatic capacity of CRC cells by enabling cell invasion and migration. In multivariate analysis, high levels of ezrin protein and mRNA in CRC samples were independent predictors of LN metastasis. Our data thus identify ezrin as a novel protein and mRNA biomarker for predicting LN metastasis in CRC patients.

Depletion of mRNA export regulator DBP5/DDX19, GLE1 or IPPK that is a key enzyme for the production of IP6, resulting in differentially altered cytoplasmic mRNA expression and specific cell defect

DBP5, also known as DDX19, GLE1 and inositol hexakisphosphate (IP6) function in messenger RNA (mRNA) export at the cytoplasmic surface of the nuclear pore complex in eukaryotic cells. DBP5 is a DEAD-box RNA helicase, and its activity is stimulated by interactions with GLE1 and IP6. In addition, these three factors also have unique role(s). To investigate how these factors influenced the cytoplasmic mRNA expression and cell phenotype change, we performed RNA microarray analysis to detect the effect and function of DBP5, GLE1 and IP6 on the cytoplasmic mRNA expression. The expression of some cytoplasmic mRNA subsets (e.g. cell cycle, DNA replication) was commonly suppressed by the knock-down of DBP5, GLE1 and IPPK (IP6 synthetic enzyme). The GLE1 knock-down selectively reduced the cytoplasmic mRNA expression required for mitotic progression, results in an abnormal spindle phenotype and caused the delay of mitotic process. Meanwhile, G1/S cell cycle arrest was observed in DBP5 and IPPK knock-down cells. Several factors that function in immune response were also down-regulated in DBP5 or IPPK knock-down cells. Thereby, IFNβ-1 mRNA transcription evoked by poly(I:C) treatment was suppressed. These results imply that DBP5, GLE1 and IP6 have a conserved and individual function in the cytoplasmic mRNA expression. Variations in phenotype are due to the difference in each function of DBP5, GLE1 and IPPK in intracellular mRNA metabolism.