Yugo Kitazawa

Recognition of floral homeotic MADS domain transcription factors by a phytoplasmal effector, phyllogen, induces phyllody.

Plant pathogens alter the course of plant developmental processes, resulting in abnormal morphology in infected host plants. Phytoplasmas are unique plant-pathogenic bacteria that transform plant floral organs into leaf-like structures and cause the emergence of secondary flowers. These distinctive symptoms have attracted considerable interest for many years. Here, we revealed the molecular mechanisms of the floral symptoms by focusing on a phytoplasma-secreted protein, PHYL1, which induces morphological changes in flowers that are similar to those seen in phytoplasma-infected plants. PHYL1 is a homolog of the phytoplasmal effector SAP54 that also alters floral development. Using yeast two-hybrid and in planta transient co-expression assays, we found that PHYL1 interacts with and degrades the floral homeotic MADS domain proteins SEPALLATA3 (SEP3), APETALA1 (AP1) and CAULIFLOWER (CAL). This degradation of MADS domain proteins was dependent on the ubiquitin–proteasome pathway. The expression of floral development genes downstream of SEP3 and AP1 was disrupted in 35S::PHYL1 transgenic plants. PHYL1 was genetically and functionally conserved among other phytoplasma strains and species. We designate PHYL1, SAP54 and their homologs as members of the phyllody-inducing gene family of ‘phyllogens’.

Identification and characterization of two novel genomic RNA segments of fig mosaic virus, RNA5 and RNA6

Fig mosaic virus (FMV), a negative-strand RNA virus, is recognized as a causal agent of fig mosaic disease. We performed RT-PCR for 14 FMV isolates collected from symptomatic fig plants in Japan and Serbia using primers corresponding to the conserved 13 nt stretches found at the termini of FMV genomic segments. The resulting simultaneous amplification of all FMV genomic segments yielded four previously identified segments of FMV and two novel segments. These novel FMV genomic RNA segments were found in each of the 14 FMV isolates analysed. In Northern blot studies, both the sense and antisense strands of these novel RNA molecules accumulated in FMV-infected fig leaves but not in uninfected fig leaves, confirming that they replicate as FMV genomic segments. Sequence analysis showed that the novel RNA segments are similar, in their structural organization and molecular evolutionary patterns, to those of known FMV genomic RNA segments. Our findings thus indicate that these newly discovered RNA segments are previously unidentified FMV genomic segments, which we have designated RNA5 and RNA6.

Rapid and reliable detection of phytoplasma by loop-mediated isothermal amplification targeting a housekeeping gene

Phytoplasmas are plant pathogenic bacteria that infect more than 700 plant species. Because phytoplasma-resistant cultivars are not available for the vast majority of crops, the most common practice to prevent phytoplasma diseases is to remove infected plants. Therefore, developing a rapid, accurate diagnostic method to detect a phytoplasma infection is important. Here, we developed a phytoplasma detection assay based on loop-mediated isothermal amplification (LAMP) by targeting the groEL gene and 16S rDNA. We designed 19 primer sets for the LAMP assay and evaluated their amplification efficiency, sensitivity, and spectra to select the most suitable primer sets to detect Candidatus Phytoplasma asteris. As a result, DNA was efficiently amplified by one of the primer sets targeting the groEL gene, and LAMP assay sensitivity with this primer set was 10-fold higher than that of the polymerase chain reaction. Moreover, the groEL gene was successfully amplified from several strains of Ca. Phytoplasma asteris by this primer set, indicating that the groEL gene can be used as a LAMP assay target gene for a broad range of phytoplasma strains. Additionally, a simple DNA extraction method that omits the homogenizing and phenol extraction steps was combined with the LAMP assay to develop a simple, rapid, and convenient diagnostic method for detecting phytoplasma.

Cell Death Triggered by a Putative Amphipathic Helix of Radish mosaic virus Helicase Protein Is Tightly Correlated With Host Membrane Modification

Systemic necrosis is one of the most severe symptoms caused by plant RNA viruses. Recently, systemic necrosis has been suggested to have similar features to a defense response referred to as the hypersensitive response (HR), a form of programmed cell death. In virus-infected plant cells, host intracellular membrane structures are changed dramatically for more efficient viral replication. However, little is known about whether this replication-associated membrane modification is the cause of the symptoms. In this study, we identified an amino-terminal amphipathic helix of the helicase encoded by Radish mosaic virus (RaMV) (genus Comovirus) as an elicitor of cell death in RaMV-infected plants. Cell death caused by the amphipathic helix had features similar to HR, such as SGT1-dependence. Mutational analyses and inhibitor assays using cerulenin demonstrated that the amphipathic helix-induced cell death was tightly correlated with dramatic alterations in endoplasmic reticulum (ER) membrane structures. Furthermore, the cell death-inducing activity of the amphipathic helix was conserved in Cowpea mosaic virus (genus Comovirus) and Tobacco ringspot virus (genus Nepovirus), both of which are classified in the family Secoviridae. Together, these results indicate that ER membrane modification associated with viral intracellular replication may be recognized to prime defense responses against plant viruses.

Purple top symptoms are associated with reduction of leaf cell death in phytoplasma-infected plants

Plants exhibit a wide variety of disease symptoms in response to pathogen attack. In general, most plant symptoms are recognized as harmful effects on host plants, and little is known about positive aspects of symptoms for infected plants. Herein, we report the beneficial role of purple top symptoms, which are characteristic of phytoplasma-infected plants. First, by using plant mutants defective in anthocyanin biosynthesis, we demonstrated that anthocyanin accumulation is directly responsible for the purple top symptoms, and is associated with reduction of leaf cell death caused by phytoplasma infection. Furthermore, we revealed that phytoplasma infection led to significant activation of the anthocyanin biosynthetic pathway and dramatic accumulation of sucrose by about 1000-fold, which can activate the anthocyanin biosynthetic pathway. This is the first study to demonstrate the role and mechanism of the purple top symptoms in plant–phytoplasma interactions.

Degradation of class E MADS-domain transcription factors in Arabidopsis by a phytoplasmal effector, phyllogen

Members of the SEPALLATA (SEP) gene sub-family encode class E floral homeotic MADS-domain transcription factors (MADS TFs) that specify the identity of floral organs. The Arabidopsis thaliana genome contains 4 ancestrally duplicated and functionally redundant SEP genes, SEP1–4. Recently, a gene family of unique effectors, phyllogens, was identified as an inducer of leaf-like floral organs in phytoplasmas (plant pathogenic bacteria). While it was shown that phyllogens target some MADS TFs, including SEP3 for degradation, it is unknown whether the other SEPs (SEP1, SEP2, and SEP4) of Arabidopsis are also degraded by them. In this study, we found that all 4 SEP proteins of Arabidopsis are degraded by a phyllogen using a transient co-expression assay in Nicotiana benthamiana. This finding indicates that phyllogens may broadly target class E MADS TFs of plants.

First report of bacterial leaf blight on cosmos (Cosmos bipinnatus Cav.) caused by Pseudomonas cichorii in Japan

Since 2006, a commercial grower in Japan has noted a leaf blight symptom on potted cosmos plants grown in a field. In August 2012, a Pseudomonas-like bacterium was isolated from the symptomatic leaves and found to cause the same symptom on cosmos seedlings after inoculation. On the basis of bacteriological and phylogenetic analyses, the causative bacterium was identified as Pseudomonas cichorii. This is the first report of bacterial leaf blight on cosmos caused by P. cichorii in Japan.

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

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 (MPFMV) using live cell imaging, focusing on its ER-directing signal peptide (SPFMV). Transiently expressed MPFMV was distributed predominantly in PD and patchy microdomains of the PM. Investigation of ER translocation efficiency revealed that SPFMV has quite low efficiency compared with SPs of well-characterized plant proteins, calreticulin and CLAVATA3. An MPFMV mutant lacking SPFMV localized exclusively to the PM microdomains, whereas SP chimeras, in which the SP of MPFMV was replaced by an SP of calreticulin or CLAVATA3, localized exclusively to the nodes of the ER, which was labeled with Arabidopsis synaptotagmin 1, a major component of ER-PM contact sites. From these results, we speculated that the low translocation efficiency of SPFMV 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 MPFMV 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 MPFMV that fails to be translocated is transferred to the microdomains, whereas the remainder of MPFMV 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 MPFMV into PD.

Functional characterization of the principal sigma factor RpoD of phytoplasmas via an in vitro transcription assay

Phytoplasmas (class, Mollicutes) are insect-transmissible and plant-pathogenic bacteria that multiply intracellularly in both plants and insects through host switching. Our previous study revealed that phytoplasmal sigma factor rpoD of OY-M strain (rpoDOY) could be a key regulator of host switching, because the expression level of rpoDOY was higher in insect hosts than in plant hosts. In this study, we developed an in vitro transcription assay system to identify RpoDOY-dependent genes and the consensus promoter elements. The assay revealed that RpoDOY regulated some housekeeping, virulence, and host–phytoplasma interaction genes of OY-M strain. The upstream region of the transcription start sites of these genes contained conserved –35 and –10 promoter sequences, which were similar to the typical bacterial RpoD-dependent promoter elements, while the –35 promoter elements were variable. In addition, we searched putative RpoD-dependent genes based on these promoter elements on the whole genome sequence of phytoplasmas using in silico tools. The phytoplasmal RpoD seems to mediate the transcription of not only many housekeeping genes as the principal sigma factor, but also the virulence- and host-phytoplasma interaction-related genes exhibiting host-specific expression patterns. These results indicate that more complex mechanisms exist than previously thought regarding gene regulation enabling phytoplasmas to switch hosts.

Construction of an infectious cDNA clone of radish mosaic virus, a crucifer-infecting comovirus.

Radish mosaic virus (RaMV) is a crucifer-infecting comovirus that has been detected worldwide. Here, we report the successful construction of a full-length infectious cDNA clone of RaMV. The full-length cDNA clones corresponding to RNA1 and RNA2 of a Japanese isolate of RaMV were cloned into the pBlueScript plasmid or the binary vector pCAMBIA1301 downstream of the cauliflower mosaic virus 35S promoter. Mechanical inoculation or agroinoculation of Nicotiana benthamiana with these vectors resulted in systemic RaMV infections causing symptoms similar to those caused by the wild-type parental virus. The presence of progeny virus was verified by western blot analysis and electron microscopy.