Phytoplasma-conserved phyllogen proteins induce phyllody across the Plantae by degrading floral MADS domain proteins.

ABCE-class MADS domain transcription factors (MTFs) are key regulators of floral organ development in angiosperms. Aberrant expression of these genes can result in abnormal floral traits such as phyllody. Phyllogen is a virulence factor conserved in phytoplasmas, plant pathogenic bacteria of the class Mollicutes. It triggers phyllody in Arabidopsis thaliana by inducing degradation of A- and E-class MTFs. However, it is still unknown whether phyllogen can induce phyllody in plants other than A. thaliana, although phytoplasma-associated phyllody symptoms are observed in a broad range of angiosperms. In this study, phyllogen was shown to cause phyllody phenotypes in several eudicot species belonging to three different families. Moreover, phyllogen can interact with MTFs of not only angiosperm species including eudicots and monocots but also gymnosperms and a fern, and induce their degradation. These results suggest that phyllogen induces phyllody in angiosperms and inhibits MTF function in diverse plant species.

EXA1, a GYF domain protein, is responsible for loss-of-susceptibility to plantago asiatica mosaic virus in Arabidopsis thaliana.

One of the plant host resistance machineries to viruses is attributed to recessive alleles of genes encoding critical host factors for virus infection. This type of resistance, also referred to as recessive resistance, is useful for revealing plant-virus interactions and for breeding antivirus resistance in crop plants. Therefore, it is important to identify a novel host factor responsible for robust recessive resistance to plant viruses. Here, we identified a mutant from an ethylmethane sulfonate (EMS)-mutagenized Arabidopsis population which confers resistance to plantago asiatica mosaic virus (PlAMV, genus Potexvirus). Based on map-based cloning and single nucleotide polymorphism analysis, we identified a premature termination codon in a functionally unknown gene containing a GYF domain, which binds to proline-rich sequences in eukaryotes. Complementation analyses and robust resistance to PlAMV in a T-DNA mutant demonstrated that this gene, named Essential for poteXvirus Accumulation 1 (EXA1), is indispensable for PlAMV infection. EXA1 contains a GYF domain and a conserved motif for interaction with eukaryotic translation initiation factor 4E (eIF4E), and is highly conserved among monocot and dicot species. Analysis using qRT-PCR and immunoblotting revealed that EXA1 was expressed in all tissues, and was not transcriptionally responsive to PlAMV infection in Arabidopsis plants. Moreover, accumulation of PlAMV and a PlAMV-derived replicon was drastically diminished in the initially infected cells by the EXA1 deficiency. Accumulation of two other potexviruses also decreased in exa1-1 mutant plants. Our results provided a functional annotation to GYF domain-containing proteins by revealing the function of the highly conserved EXA1 gene in plant-virus 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.

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.

The phytoplasmal virulence factor TENGU causes plant sterility by downregulating of the jasmonic acid and auxin pathways.

Despite plants infected by pathogens are often unable to produce offspring, it remains unclear how sterility is induced in host plants. In this study, we demonstrate that TENGU, a phytoplasmal virulence peptide known as a dwarfism inducer, acts as an inducer of sterility. Transgenic expression of TENGU induced both male and female sterility in Arabidopsis thaliana flowers similar to those observed in double knockout mutants of auxin response factor 6 (ARF6) and ARF8, which are known to regulate floral development in a jasmonic acid (JA)-dependent manner. Transcripts of ARF6 and ARF8 were significantly decreased in both tengu-transgenic and phytoplasma-infected plants. Furthermore, JA and auxin levels were actually decreased in tengu-transgenic buds, suggesting that TENGU reduces the endogenous levels of phytohormones by repressing ARF6 and ARF8, resulting in impaired flower maturation. TENGU is the first virulence factor with the effects on plant reproduction by perturbation of phytohormone signaling.

Onion yellow phytoplasma P38 protein plays a role in adhesion to the hosts.

Adhesins are microbial surface proteins that mediate the adherence of microbial pathogens to host cell surfaces. In Mollicutes, several adhesins have been reported in mycoplasmas and spiroplasmas. Adhesins P40 of Mycoplasma agalactiae and P89 of Spiroplasma citri contain a conserved amino acid sequence known as the Mollicutes adhesin motif (MAM), whose function in the host cell adhesion remains unclear. Here, we show that phytoplasmas, which are plant-pathogenic mollicutes transmitted by insect vectors, possess an adhesion-containing MAM that was identified in a putative membrane protein, PAM289 (P38), of the 'Candidatus Phytoplasma asteris,' OY strain. P38 homologs and their MAMs were highly conserved in related phytoplasma strains. While P38 protein was expressed in OY-infected insect and plant hosts, binding assays showed that P38 interacts with insect extract, and weakly with plant extract. Interestingly, the interaction of P38 with the insect extract depended on MAM. These results suggest that P38 is a phytoplasma adhesin that interacts with the hosts. In addition, the MAM of adhesins is important for the interaction between P38 protein and hosts.

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'.

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.

The alteration of plant morphology by small peptides released from the proteolytic processing of the bacterial peptide TENGU.

Phytoplasmas are insect-borne plant pathogenic bacteria that alter host morphology. TENGU, a small peptide of 38 residues, is a virulence factor secreted by phytoplasmas that induces dwarfism and witches' broom in the host plant. In this study, we demonstrate that plants process TENGU in order to generate small functional peptides. First, virus vector-mediated transient expression demonstrated that the amino-terminal 11 amino acids of TENGU are capable of causing symptom development in Nicotiana benthamiana plants. The deletion of the 11th residue significantly diminished the symptom-inducing activity of TENGU, suggesting that these 11 amino acids constitute a functional domain. Second, we found that TENGU undergoes proteolytic processing in vitro, generating peptides of 19 and 21 residues including the functional domain. Third, we observed similar processing of TENGU in planta, and an alanine substitution mutant of TENGU, for which processing was compromised, showed reduced symptom induction activity. All TENGU homologs from several phytoplasma strains possessed similar symptom induction activity and went through processing, which suggests that the processing of TENGU might be related to its function.

Functional characterization and gene expression profiling of superoxide dismutase from plant pathogenic phytoplasma.

The rapid production of huge amounts of reactive oxygen species (ROS) is one of the responses of animal and plant cells induced under stress conditions, such as pathogenic bacterial infection. To protect against the cytotoxic ROS, it is important for pathogenic bacteria to inactivate ROS by employing their antioxidant enzymes like superoxide dismutase (SOD). Here, we cloned and characterized the sodA gene from the plant pathogenic bacterium, 'Candidatus Phytoplasma asteris' OY-W strain. This is the first description of gene expression and antioxidant enzymatic activity of SOD from a phytoplasma. We also demonstrated the sodA gene product (OY-SOD) functions as Mn-type SOD. Since other Mollicutes bacteria such as mycoplasmas do not possess sodA probably due to reductive evolution, it is intriguing that phytoplasmas possess sodA despite their lack of many metabolic genes, suggesting that OY-SOD may play an important role in the phytoplasma colonization of plants and insects. Moreover, Western blot analysis and real-time PCR revealed that OY-SOD is expressed when the phytoplasma is grown in both plant and insect hosts, suggesting it is functioning in both hosts. Possible role of SOD in protection against damage by host-derived ROS is discussed.

Lectin-mediated resistance impairs plant virus infection at the cellular level.

Plants possess a multilayered defense response, known as plant innate immunity, to infection by a wide variety of pathogens. Lectins, sugar binding proteins, play essential roles in the innate immunity of animal cells, but the role of lectins in plant defense is not clear. This study analyzed the resistance of certain Arabidopsis thaliana ecotypes to a potexvirus, plantago asiatica mosaic virus (PlAMV). Map-based positional cloning revealed that the lectin gene JACALIN-TYPE LECTIN REQUIRED FOR POTEXVIRUS RESISTANCE1 (JAX1) is responsible for the resistance. JAX1-mediated resistance did not show the properties of conventional resistance (R) protein-mediated resistance and was independent of plant defense hormone signaling. Heterologous expression of JAX1 in Nicotiana benthamiana showed that JAX1 interferes with infection by other tested potexviruses but not with plant viruses from different genera, indicating the broad but specific resistance to potexviruses conferred by JAX1. In contrast with the lectin gene RESTRICTED TEV MOVEMENT1, which inhibits the systemic movement of potyviruses, which are distantly related to potexviruses, JAX1 impairs the accumulation of PlAMV RNA at the cellular level. The existence of lectin genes that show a variety of levels of virus resistance, their targets, and their properties, which are distinct from those of known R genes, suggests the generality of lectin-mediated resistance in plant innate immunity.

Dramatic transcriptional changes in an intracellular parasite enable host switching between plant and insect.

Phytoplasmas are bacterial plant pathogens that have devastating effects on the yields of crops and plants worldwide. They are intracellular parasites of both plants and insects, and are spread among plants by insects. How phytoplasmas can adapt to two diverse environments is of considerable interest; however, the mechanisms enabling the "host switching" between plant and insect hosts are poorly understood. Here, we report that phytoplasmas dramatically alter their gene expression in response to "host switching" between plant and insect. We performed a detailed characterization of the dramatic change that occurs in the gene expression profile of Candidatus Phytoplasma asteris OY-M strain (approximately 33% of the genes change) upon host switching between plant and insect. The phytoplasma may use transporters, secreted proteins, and metabolic enzymes in a host-specific manner. As phytoplasmas reside within the host cell, the proteins secreted from phytoplasmas are thought to play crucial roles in the interplay between phytoplasmas and host cells. Our microarray analysis revealed that the expression of the gene encoding the secreted protein PAM486 was highly upregulated in the plant host, which is also observed by immunohistochemical analysis, suggesting that this protein functions mainly when the phytoplasma grows in the plant host. Additionally, phytoplasma growth in planta was partially suppressed by an inhibitor of the MscL osmotic channel that is highly expressed in the plant host, suggesting that the osmotic channel might play an important role in survival in the plant host. These results also suggest that the elucidation of "host switching" mechanism may contribute to the development of novel pest controls.

Unique morphological changes in plant pathogenic phytoplasma-infected petunia flowers are related to transcriptional regulation of floral homeotic genes in an organ-specific manner.

Abnormal flowers are often induced by infection of certain plant pathogens, e.g. phytoplasma, but the molecular mechanisms underlying these malformations have remained poorly understood. Here, we show that infection with OY-W phytoplasma (Candidatus Phytoplasma asteris, onion yellows phytoplasma strain, line OY-W) affects the expression of the floral homeotic genes of petunia plants in an organ-specific manner. Upon infection with OY-W phytoplasma, floral morphological changes, including conversion to leaf-like structures, were observed in sepals, petals and pistils, but not in stamens. As the expression levels of homeotic genes differ greatly between floral organs, we examined the expression levels of homeotic genes in each floral organ infected by OY-W phytoplasma, compared with healthy plants. The expression levels of several homeotic genes required for organ development, such as PFG, PhGLO1 and FBP7, were significantly downregulated by the phytoplasma infection in floral organs, except the stamens, suggesting that the unique morphological changes caused by the phytoplasma infection might result from the significant decrease in expression of some crucial homeotic genes. Moreover, the expression levels of TER, ALF and DOT genes, which are known to participate in floral meristem identity, were significantly downregulated in the phytoplasma-infected petunia meristems, implying that phytoplasma would affect an upstream signaling pathway of floral meristem identity. Our results suggest that phytoplasma infection may have complex effects on floral development, resulting in the unique phenotypes that were clearly distinct from the mutant flower phenotypes produced by the knock-out or the overexpression of certain homeotic genes.

Molecular epidemiology of Plum pox virus in Japan.

For a molecular epidemiological study based on complete genome sequences, 37 Plum pox virus (PPV) isolates were collected from the Kanto region in Japan. Pair-wise analyses revealed that all 37 Japanese isolates belong to the PPV-D strain, with low genetic diversity (less than 0.8%). In phylogenetic analysis of the PPV-D strain based on complete nucleotide sequences, the relationships of the PPV-D strain were reconstructed with high resolution: at the global level, the American, Canadian, and Japanese isolates formed their own distinct monophyletic clusters, suggesting that the routes of viral entry into these countries were independent; at the local level, the actual transmission histories of PPV were precisely reconstructed with high bootstrap support. This is the first description of the molecular epidemiology of PPV based on complete genome sequences.

Viral-induced systemic necrosis in plants involves both programmed cell death and the inhibition of viral multiplication, which are regulated by independent pathways.

Resistant plants respond rapidly to invading avirulent plant viruses by triggering a hypersensitive response (HR). An HR is accompanied by a restraint of virus multiplication and programmed cell death (PCD), both of which have been observed in systemic necrosis triggered by a successful viral infection. Here, we analyzed signaling pathways underlying the HR in resistance genotype plants and those leading to systemic necrosis. We show that systemic necrosis in Nicotiana benthamiana, induced by Plantago asiatica mosaic virus (PlAMV) infection, was associated with PCD, biochemical features, and gene expression patterns that are characteristic of HR. The induction of necrosis caused by PlAMV infection was dependent on SGT1, RAR1, and the downstream mitogen-activated protein kinase (MAPK) cascade involving MAPKKKalpha and MEK2. However, although SGT1 and RAR1 silencing led to an increased accumulation of PlAMV, silencing of the MAPKKKalpha-MEK2 cascade did not. This observation indicates that viral multiplication is partly restrained even in systemic necrosis induced by viral infection, and that this restraint requires SGT1 and RAR1 but not the MAPKKKalpha-MEK2 cascade. Similarly, although both SGT1 and MAPKKKalpha were essential for the Rx-mediated HR to Potato virus X (PVX), SGT1 but not MAPKKKalpha was involved in the restraint of PVX multiplication. These results suggest that systemic necrosis and HR consist of PCD and a restraint of virus multiplication, and that the latter is induced through unknown pathways independent from the former.

Significantly low level of small RNA accumulation derived from an encapsidated mycovirus with dsRNA genome.

The role of RNA silencing as an antiviral defence has been well elucidated in plants and invertebrates, but not in filamentous fungi. We have previously determined the complete genome sequence of Magnaporthe oryzae virus 2 (MoV2), a dsRNA virus that infects the rice blast fungus Magnaporthe oryzae. In this study, we detected small interfering RNAs (siRNAs) from both positive- and negative-strand MoV2 viral RNA, suggesting that the RNA silencing machinery in M. oryzae functions against the mycovirus. Cloning and characterisation of MoV2 siRNAs indicated that, in MoV2, the ratio of virus-derived siRNAs to total small RNA is significantly lower than that in either plant viruses or Cryphonectria hypovirus 1 (CHV1), another mycovirus. Nevertheless, any MoV2-encoded proteins did not exhibit RNA silencing suppressor activity in both the plant and fungal systems. Our study suggests the existence of a novel viral strategy employed to evade host RNA silencing.

The N-terminal region of the Plantago asiatica mosaic virus coat protein is required for cell-to-cell movement but is dispensable for virion assembly.

Potexvirus cell-to-cell movement requires coat protein (CP) and movement proteins. In this study, mutations in two conserved in-frame AUG codons in the 5' region of the CP open reading frame of Plantago asiatica mosaic virus (PlAMV) were introduced, and virus accumulation of these mutants was analyzed in inoculated and upper noninoculated leaves. When CP was translated only from the second AUG codon, virus accumulation in inoculated leaves was lower than that of wild-type PlAMV, and the viral spread was impaired. Trans-complementation analysis showed that the leucine residue at the third position (Leu-3) of CP is important for cell-to-cell movement of PlAMV. The 14-amino-acid N-terminal region of CP was dispensable for virion formation. Immunoprecipitation assays conducted with an anti-TGBp1 antibody indicated that PlAMV CP interacts with TGBp1 in vivo and that this interaction is not affected by alanine substitution at Leu-3. These results support the concept that the N-terminal region of potexvirus CP can be separated into two distinct functional domains.

Magnaporthe oryzae endopolygalacturonase homolog correlates with density-dependent conidial germination.

Endopolygalacturonases (endoPGs) of some phytopathogens are virulent factors for dicots. To investigate the function of the endoPG of Magnaporthe oryzae, a disruption mutant of MGG_08938, the homolog of endoPG found in the genome database of this fungus, was generated. The pathogenicity, mycelial growth, and appressorium formation of this mutant were comparable with those of the wild-type strain; however, the germination of conidia in a highly concentrated suspension of conidia was affected by the mutation. Whereas the germination of the wild-type strain was inhibited at high concentrations, this effect was canceled out by disruption by the endoPG homolog gene. The authors named the gene MDG1 (M. oryzae density-dependent germination), which delineates this new function in the fungus.