Misako Himeno

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.

Lectin-Mediated Resistance Impairs Plant Virus Infection at the Cellular Level

This work identifies jacalin-type lectin that is responsible for resistance to multiple plant viruses belonging to the genus Potexvirus. The isolation and characterization of this lectin sheds light on a novel resistance machinery to plant viruses. 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.

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’.

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.

Complete nucleotide sequence of a new double-stranded RNA virus from the rice blast fungus, Magnaporthe oryzae

Magnaporthe oryzae B. Couch (formerly Magnaporthe grisea (Hebert) Barr) [3] is the causal agent of the devastating rice blast disease [6]. This filamentous fungus is being studied intensively as a principal model organism for understanding plantfungus interactions. Virus-like particles (VLPs) were previously detected in two M. oryzae strains (TH 65-105 and Ken 60-19) using electron microscopy [8]. Recently, the virus isolated from M. oryzae strain TH 65-105 was designated Magnaporthe oryzae virus 1 (MoV1) [9]. In this study, we isolated an additional M. oryzae virus from another strain, Ken 60-19. This virus has a different gene organization to MoV1. The virus particles are small spheres, approximately 37 nm in diameter, containing non-segmented dsRNA genomes of 5.2 kbp, both of which are features of members of the family Totiviridae. Sequence comparisons and phylogenetic analyses revealed that the virus isolated from M. oryzae strain Ken 60-19 is closely related to viruses of the family Totiviridae infecting filamentous fungi.

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

A bacterial peptide effector undergoes proteolytic processing in plants and releases small peptides that alter plant morphology. 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.

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.

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.

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.