Analysis of Plant RRP44/DIS3, a component of the RNA Exosome

学位の種別:課程博士University of Tokyo(東京大学

Comparative transient expression analyses on two conserved effectors of Colletotrichum orbiculare reveal their distinct cell death‐inducing activities between Nicotiana benthamiana and melon

Colletotrichum orbiculare infects cucurbits, such as cucumber and melon (Cucumis melo), as well as the model Solanaceae plant Nicotiana benthamiana, by secreting an arsenal of effectors that suppress the immunity of these distinct plants. Two conserved effectors of C. orbiculare, called NLP1 and NIS1, induce cell death responses in N. benthamiana, but it is unclear whether they exhibit the same activity in Cucurbitaceae plants. In this study, we established a new Agrobacterium-mediated transient expression system to investigate the cell death-inducing activity of NLP1 and NIS1 in melon. NLP1 strongly induced cell death in melon but, in contrast to the effects seen in N. benthamiana, mutations either in the heptapeptide motif or in the putative glycosylinositol phosphorylceramide-binding site did not cancel its cell death-inducing activity in melon. Furthermore, NLP1 lacking the signal peptide caused cell death in melon but not in N. benthamiana. Study of the transient expression of NIS1 also revealed that, unlike in N. benthamiana, NIS1 did not induce cell death in melon. In contrast, NIS1 suppressed flg22-induced reactive oxygen species generation in melon, as seen in N. benthamiana. These findings indicate distinct cell death-inducing activities of NLP1 and NIS1 in these two plant species that C. orbiculare infects

Regulation of High-Temperature Stress Response by Small RNAs

Temperature extremes constitute one of the most common environmental stresses that adversely affect the growth and development of plants. Transcriptional regulation of temperature stress responses, particularly involving protein-coding gene networks, has been intensively studied in recent years. High-throughput sequencing technologies enabled the detection of a great number of small RNAs that have been found to change during and following temperature stress. The precise molecular action of some of these has been elucidated in detail. In the present chapter, we summarize the current understanding of small RNA-mediated modulation of high- temperature stress-regulatory pathways including basal stress responses, acclimation, and thermo-memory. We gather evidence that suggests that small RNA network changes, involving multiple upregulated and downregulated small RNAs, balance the trade-off between growth/development and stress responses, in order to ensure successful adaptation. We highlight specific characteristics of small RNA-based tem- perature stress regulation in crop plants. Finally, we explore the perspectives of the use of small RNAs in breeding to improve stress tolerance, which may be relevant for agriculture in the near future

Genus-Wide Comparative Genome Analyses of Colletotrichum Species Reveal Specific Gene Family Losses and Gains during Adaptation to Specific Infection Lifestyles.

Members from Colletotrichum genus adopt a diverse range of lifestyles during infection of plants and represent a group of agriculturally devastating pathogens. In this study, we present the draft genome of Colletotrichum incanum from the spaethianum clade of Colletotrichum and the comparative analyses with five other Colletotrichum species from distinct lineages. We show that the C. incanum strain, originally isolated from Japanese daikon radish, is able to infect both eudicot plants, such as certain ecotypes of the eudicot Arabidopsis, and monocot plants, such as lily. Being closely related to Colletotrichum species both in the graminicola clade, whose members are restricted strictly to monocot hosts, and to the destructivum clade, whose members are mostly associated with dicot infections, C. incanum provides an interesting model system for comparative genomics to study how fungal pathogens adapt to monocot and dicot hosts. Genus-wide comparative genome analyses reveal that Colletotrichum species have tailored profiles of their carbohydrate-degrading enzymes according to their infection lifestyles. In addition, we show evidence that positive selection acting on secreted and nuclear localized proteins that are highly conserved may be important in adaptation to specific hosts or ecological niches

Identification of novel sesterterpenes by genome mining of phytopathogenic fungi Phoma and Colletotrichum sp.

Two homologous gene clusters for the biosynthesis of sesterterpenes betaestacins were identified from two phytopathogens, Phoma betae and Colletotrichum orbiculare. Heterologous expression of identified oxidation enzymes with previously-characterized PbTS1 (BtcA(pb)) resulted in the production of seven novel sesterterpenes. Although both strains possessed homologous enzymes, oxidation state of corresponding products were different from each other, suggesting that structural diversification of sesterterpene skeletons might be achieved by these homologous enzymes with different functions. (C) 2013 Elsevier Ltd. All rights reserved

<i>Arabidopsis</i> AtRRP44A Is the Functional Homolog of Rrp44/Dis3, an Exosome Component, Is Essential for Viability and Is Required for RNA Processing and Degradation

<div><p>The RNA exosome is a multi-subunit complex that is responsible for 3ʹ to 5ʹ degradation and processing of cellular RNA. Rrp44/Dis3 is the catalytic center of the exosome in yeast and humans. However, the role of Rrp44/Dis3 homologs in plants is still unidentified. Here, we show that <i>Arabidopsis</i> AtRRP44A is the functional homolog of Rrp44/Dis3, is essential for plant viability and is required for RNA processing and degradation. We characterized AtRRP44A and AtRRP44B/SOV, two predicted <i>Arabidopsis</i> Rrp44/Dis3 homologs. AtRRP44A could functionally replace <i>S. cerevisiae</i> Rrp44/Dis3, but AtRRP44B/SOV could not. <i>rrp44a</i> knock-down mutants showed typical phenotypes of exosome function deficiency, 5.8S rRNA 3ʹ extension and rRNA maturation by-product over-accumulation, but <i>rrp44b</i> mutants did not. Conversely, AtRRP44B/SOV mutants showed elevated levels of a selected mRNA, on which <i>rrp44a</i> did not have detectable effects. Although T-DNA insertion mutants of AtRRP44B/SOV had no obvious phenotype, those of AtRRP44A showed defects in female gametophyte development and early embryogenesis. These results indicate that AtRRP44A and AtRRP44B/SOV have independent roles for RNA turnover in plants.</p> </div

Model for the roles of <i>A. thaliana</i> AtRRP44A and AtRRP44B/SOV in RNA processing and degradation.

<p>AtRRP44A localizes to the nucleus and processes rRNAs with the exosome complex. However, AtRRP44B/SOV localizes to the cytoplasm and targets a select subset of mRNAs.</p

<i>Arabidopsis</i> Rrp44/Dis3 homologs.

<p>(A) Schematics of the Rrp44/Dis3 homologs <i>S. cerevisiae</i> Rrp44 (ScRrp44), human RRP44/DIS3 (hRRP44), and <i>A. thaliana</i> AtRRP44A and AtRRP44B/SOV. Yellow and blue boxes represent the PIN and RNB domains, respectively, that are conserved among Rrp44/Dis3 homologs. aa represents amino acids. (B) AtRRP44A complements the S. <i>cerevisiae </i><i>rrp44</i> doxycycline (DOX) repressible mutant. Growth phenotypes resulting from the expression of plasmid-borne AtRRP44A, AtRRP44B/SOV, AtRRP44A and AtRRP44B/SOV, and ScRrp44 in <i>S. cerevisiae</i> BSY1883 strain, and negative control alleles were assessed in the presence (repressed chromosomal ScRrp44) or absence (expressed chromosomal ScRrp44) of DOX after incubation for 90 h at 30°C. –LEU-TRP, without leucine and tryptophan. (C) Diagram of the intron–exon structure of AtRRP44A and AtRRP44B/SOV. UTRs are indicated by grey boxes, exons by black boxes and introns by solid lines. T-DNA insertion sites for <i>rrp44a-1</i> (SALK_037533), <i>rrp44a-2</i> (SALK_141741), <i>rrp44a-3</i> (SALK_051800), <i>rrp44b-1</i> (SAIL_804_F05), <i>rrp44b-2</i> (SALK_017934) and <i>rrp44b-3</i> (SALK_010765) are shown in red arrowheads.</p

Analysis of rRNA processing and degradation.

<p>(A) Diagram illustrating the 5.8S rRNA processing intermediates and the rRNA maturation by-product generated from the 5ʹ ETS (P-Pʹ) compared with the 35S precursor [27]. Horizontal red arrows represent the positions of oligonucleotide probes used in this study. (B) The 5.8S rRNA 3ʹ extension is processed by AtRRP44A, AtRRP4 and AtRRP41, but not AtRRP44B/SOV. (C) The 5ʹ ETS is degraded by AtRRP44A, AtRRP4 and AtRRP41, but not AtRRP44B/SOV. RNA gel blots of 5.8S rRNA precursors (B) or the 5ʹ ETS (C). Total RNAs were isolated from 10 dpg rosette leaves of Col-0 (wild type: WT), <i>gusKD-2</i> (VC), <i>rrp44aKD-1</i>, <i>rrp44aKD-2</i>, <i>rrp44b-1</i>, <i>rrp44b-2</i> and <i>mtr4-1</i> plants or from <i>gusKD-2</i>, <i>rrp4KD-3</i>, <i>rrp41KD-1</i> and <i>rrp44aKD-1</i> plants (B and C). <i>mtr4-1</i> was used to determine the sequence of 5.8S processing intermediates [27]. Total RNAs were separated on 6% polyacrylamide gels. Methylene blue staining of 5S rRNA is shown as a loading control. Relative RNA levels estimated from band signals are indicated at the bottom of each lane as mean values ± SE with RNA levels in Col-0 plants set to 1.0.. Values for which P<0.05 (Tukey’s test) compared to corresponding wild type plants (<i>gusKD-2</i> or Col-0) were shown in red. Two (B and C: Left panels) or three (B and C: Right panels) biological replicates were performed for all RNA gel blots. </p

Levels of MRP RNA and snoRNA31 in <i>rrp44aKD-1</i>, <i>rrp44b-2</i> and the exosome core mutants.

<p>(A and B) qRT-PCR revealed that accumulation of the MRP RNA and snoRNA31 was upregulated in <i>rrp4KD-3</i>, <i>rrp41KD-1</i> and <i>rrp44aKD-1</i>, but not in <i>rrp44b-1</i>. AtRRP4 and AtRRP41 represent the <i>Arabidopsis</i> exosome core. Total RNAs were isolated from 10 dpg rosette leaves. EF1a mRNA was used as an endogenous control. Error bars represent standard errors. Three biological replicates and two technical replicates were performed. * indicates significant difference (<i>p</i> < 0.05, Tukey’s test) between mutant and wild type plants. </p