植物病原細菌の花き類に対する病原性に関する研究

学位の種別: 課程博士審査委員会委員 : （主査）東京大学教授 難波 成任, 東京大学教授 柴田 道夫, 東京大学准教授 伊藤 純一, 東京大学准教授 山次 康幸, 法政大学教授 大島 研郎University of Tokyo(東京大学

A Reverse-Transcription Loop-Mediated Isothermal Amplification Technique to Detect Tomato Mottle Mosaic Virus, an Emerging Tobamovirus

Tomato mottle mosaic virus (ToMMV) is an emerging seed-transmissible tobamovirus that infects tomato and pepper. Since the first report in 2013 in Mexico, ToMMV has spread worldwide, posing a serious threat to the production of both crops. To prevent the spread of this virus, early and accurate detection of infection is required. In this study, we developed a detection method for ToMMV based on reverse-transcription loop-mediated isothermal amplification (RT-LAMP). A LAMP primer set was designed to target the genomic region spanning the movement protein and coat protein genes, which is a highly conserved sequence unique to ToMMV. This RT-LAMP detection method achieved 10-fold higher sensitivity than conventional RT-polymerase chain reaction methods and obtained high specificity without false positives for closely related tobamoviruses or healthy tomato plants. This method can detect ToMMV within 30 min of direct sampling of an infected tomato leaf using a toothpick and therefore does not require RNA purification. Given its high sensitivity, specificity, simplicity, and rapidity, the RT-LAMP method developed in this study is expected to be valuable for point-of-care testing in field surveys and for large-scale testing

Potential mobile units drive the horizontal transfer of phytoplasma effector phyllogen genes

Phytoplasmas are obligate intracellular plant pathogenic bacteria that can induce phyllody, which is a type of abnormal floral organ development. Phytoplasmas possess phyllogens, which are effector proteins that cause phyllody in plants. Phylogenetic comparisons of phyllogen and 16S rRNA genes have suggested that phyllogen genes undergo horizontal transfer between phytoplasma species and strains. However, the mechanisms and evolutionary implications of this horizontal gene transfer are unclear. Here, we analyzed synteny in phyllogen flanking genomic regions from 17 phytoplasma strains that were related to six ‘Candidatus’ species, including three strains newly sequenced in this study. Many of the phyllogens were flanked by multicopy genes within potential mobile units (PMUs), which are putative transposable elements found in phytoplasmas. The multicopy genes exhibited two distinct patterns of synteny that correlated with the linked phyllogens. The low level of sequence identities and partial truncations found among these phyllogen flanking genes indicate that the PMU sequences are deteriorating, whereas the highly conserved sequences and functions (e.g., inducing phyllody) of the phyllogens suggest that the latter are important for phytoplasma fitness. Furthermore, although their phyllogens were similar, PMUs in strains related to ‘Ca. P. asteris’ were often located in different regions of the genome. These findings strongly indicate that PMUs drive the horizontal transfer of phyllogens among phytoplasma species and strains. These insights improve our understanding of how symptom-determinant genes have been shared among phytoplasmas

Complete Genome Sequence of the First Isolate of Hibiscus Latent Singapore Virus Detected in Japan

The complete genome sequence of the first Japanese isolate of hibiscus latent Singapore virus (HLSV-J) was determined. The genomes of HLSV-J and a reported isolate from Singapore had only 86.7% nucleotide identity, while the encoded proteins shared amino acid identities of more than 95%

Dual targeting of a virus movement protein to ER and plasma membrane subdomains is essential for plasmodesmata localization

<div><p>Plant virus movement proteins (MPs) localize to plasmodesmata (PD) to facilitate virus cell-to-cell movement. Numerous studies have suggested that MPs use a pathway either through the ER or through the plasma membrane (PM). Furthermore, recent studies reported that ER-PM contact sites and PM microdomains, which are subdomains found in the ER and PM, are involved in virus cell-to-cell movement. However, functional relationship of these subdomains in MP traffic to PD has not been described previously. We demonstrate here the intracellular trafficking of fig mosaic virus MP (MP_FMV) using live cell imaging, focusing on its ER-directing signal peptide (SP_FMV). Transiently expressed MP_FMV was distributed predominantly in PD and patchy microdomains of the PM. Investigation of ER translocation efficiency revealed that SP_FMV has quite low efficiency compared with SPs of well-characterized plant proteins, calreticulin and CLAVATA3. An MP_FMV mutant lacking SP_FMV localized exclusively to the PM microdomains, whereas SP chimeras, in which the SP of MP_FMV was replaced by an SP of calreticulin or CLAVATA3, localized exclusively to the nodes of the ER, which was labeled with <i>Arabidopsis</i> synaptotagmin 1, a major component of ER-PM contact sites. From these results, we speculated that the low translocation efficiency of SP_FMV contributes to the generation of ER-translocated and the microdomain-localized populations, both of which are necessary for PD localization. Consistent with this hypothesis, SP-deficient MP_FMV became localized to PD when co-expressed with an SP chimera. Here we propose a new model for the intracellular trafficking of a viral MP. A substantial portion of MP_FMV that fails to be translocated is transferred to the microdomains, whereas the remainder of MP_FMV that is successfully translocated into the ER subsequently localizes to ER-PM contact sites and plays an important role in the entry of the microdomain-localized MP_FMV into PD.</p></div

A model for the intracellular trafficking of MP_FMV.

<p>A model for the intracellular trafficking of MP_FMV.</p

Investigation of the subcellular distribution of MP_FMV:YFP.

<p>(A–C) Confocal imaging of MP_FMV:YFP-expressing epidermal cells at 36 hours post-infiltration (hpi). YFP fluorescence was pseudocolored by magenta. (A) (i) Cells expressing MP_FMV:YFP were treated with aniline blue. Arrowheads indicate plasmodesma. The mean ± SD of Pearson correlation coefficient (PCC [–1:1]) is given in the image. Bars = 5 μm. (ii) Fluorescence intensity along the arrow across plasmodesma. The dotted line in the confocal image indicates the cell wall. Bar = 1 μm. (B) Plasmolyzed cells expressing (i) MP_FMV:YFP or (ii) 30K:GFP. Arrows indicate Hechtian strands extended from the PM. Arrowheads indicate plasmodesmata. The dotted line indicates the cell wall. cw, cell wall; cyt, cytoplasm. Bars = 10 μm. (C) Surface views of cells (i) expressing MP_FMV:YFP or (ii) treated with FM4-64. (iii) Co-expression of MP_FMV:CFP and YFP:REM1.3. To obtain higher resolution images, images were processed by a deconvolution algorithm. Bars = 5 μm. (D) 1% TritonX-100 treatment of membranes. Membrane-enriched fractions prepared from FMV-infected fig leaves and <i>N</i>. <i>benthamiana</i> leaves expressing MP_FMV:FLAG or YFP:REM1.3 at 36 hpi were treated with 1% TritonX-100. Anti-FLAG, anti-MP_FMV, anti-GFP, anti-H⁺ATPase and anti-BIP antibodies were used for the detection of MP_FMV:FLAG, MP_FMV, YFP:REM1.3, a PM marker H⁺ATPase and an ER marker BIP. S; soluble fraction. P; insoluble fraction.</p

ER-translocated MP_FMV specifically localizes to ER-PM contact sites.

<p>(A and B) ER-translocated MP_FMV co-localized with SYTA, a protein localized to ER-PM contact sites. 3D-projection images of cells expressing SYTA:CFP and (A) SP_clvTrun:YFP or (B) MP_FMV:YFP. Z-section images of 10 slices at 1.0 μm intervals were processed. Arrowheads indicate co-localization of SYTA:CFP and MP_FMV:YFP. (C and D) Expression of SYTA^ΔC2B, a dominant-negative form of SYTA, affected MP_FMV localization. (C) SYTA^ΔC2B:myc was co-expressed with MP_FMV:YFP. Plasmodesmata were visualized by aniline blue treatment. (D) SYTA^ΔC2B:myc was co-expressed with MP_FMV:YFP and SYTA:CFP. A cortical region was visualized. (A–D) Cells were observed at 36 hpi. YFP fluorescence was pseudocolored with magenta. Bars: (A–D), 5 μm; (C) inset 2.5 μm. (E) SYTA^ΔC2B:myc showed an inhibitory effect on MP_FMV movement to adjacent cells. MP_FMV:YFP was co-expressed with GUS or SYTA^ΔC2B:myc. (i) Typical images were captured at 3 dpi. Bars = 50 μm. (ii) Quantitative analysis. The bars show means + SD. n indicates the total number of total measurements in two independent experiments. The asterisk above an error bar indicates a statistical difference at the 1% level of significance (Student's t-test). (iii) Immunoblot analysis using anti-myc antibody (top panel). CBB staining is shown as a loading control (bottom panel). Samples were collected at 36 hpi.</p