Live imaging of primary ocular vasculature formation in zebrafish.

Ocular vasculature consists of the central retinal and ciliary vascular systems, which are essential to maintaining visual function. Many researchers have attempted to determine their origins and development; however, the detailed, stepwise process of ocular vasculature formation has not been established. In zebrafish, two angioblast clusters, the rostral and midbrain organizing centers, form almost all of the cranial vasculature, including the ocular vasculature, and these are from where the cerebral arterial and venous angioblast clusters, respectively, differentiate. In this study, we first determined the anatomical architecture of the primary ocular vasculature and then followed its path from the two cerebral angioblast clusters using a time-lapse analysis of living Tg(flk1:EGFP)k7 zebrafish embryos, in which the endothelial cells specifically expressed enhanced green fluorescent protein. We succeeded in capturing images of the primary ocular vasculature formation and were able to determine the origin of each ocular vessel. In zebrafish, the hyaloid and ciliary arterial systems first organized independently, and then anastomosed via the inner optic circle on the surface of the lens by the lateral transfer of the optic vein. Finally, the choroidal vascular plexus formed around the eyeball to complete the primary ocular vasculature formation. To our knowledge, this study is the first to report successful capture of circular integration of the optic artery and vein, lateral transfer of the optic vein to integrate the hyaloidal and superficial ocular vasculatures, and formation of the choroidal vascular plexus. Furthermore, this new morphological information enables us to assess the entire process of the primary ocular vasculature formation, which will be useful for its precise understanding.

MoST1 encoding a hexose transporter-like protein is involved in both conidiation and mycelial melanization of Magnaporthe oryzae.

In a large-scale gene disruption screen of Magnaporthe oryzae, a gene MoST1 encoding a protein belonging to the hexose transporter family was identified as a gene required for conidiation and culture pigmentation. The gene MoST1 located on chromosome V of the M. oryzae genome was predicted to be 1892 bp in length with two introns encoding a 547-amino-acid protein with 12 putative transmembrane domains. Targeted gene disruption of MoST1 resulted in a mutant (most1) with extremely poor conidiation and defects in colony melanization. These phenotypes were complemented by re-introduction of an intact copy of MoST1. We generated a transgenic line harboring a vector containing the MoST1 promoter fused with a reporter protein gene mCherry. The mCherry fluorescence was observed in mycelia, conidia, germ tubes, and appressoria in M. oryzae. There are 66 other hexose transporter-like genes in M. oryzae, and we performed complementation assay with three genes most closely related to MoST1. However, none of them complemented the most1 mutant in conidiation and melanization, indicating that the homologs do not complement the function of MoST1. These results suggest that MoST1 has a specific role for conidiation and mycelial melanization, which is not shared by other hexose transporter family of M. oryzae.

Large-scale gene disruption in Magnaporthe oryzae identifies MC69, a secreted protein required for infection by monocot and dicot fungal pathogens.

To search for virulence effector genes of the rice blast fungus, Magnaporthe oryzae, we carried out a large-scale targeted disruption of genes for 78 putative secreted proteins that are expressed during the early stages of infection of M. oryzae. Disruption of the majority of genes did not affect growth, conidiation, or pathogenicity of M. oryzae. One exception was the gene MC69. The mc69 mutant showed a severe reduction in blast symptoms on rice and barley, indicating the importance of MC69 for pathogenicity of M. oryzae. The mc69 mutant did not exhibit changes in saprophytic growth and conidiation. Microscopic analysis of infection behavior in the mc69 mutant revealed that MC69 is dispensable for appressorium formation. However, mc69 mutant failed to develop invasive hyphae after appressorium formation in rice leaf sheath, indicating a critical role of MC69 in interaction with host plants. MC69 encodes a hypothetical 54 amino acids protein with a signal peptide. Live-cell imaging suggested that fluorescently labeled MC69 was not translocated into rice cytoplasm. Site-directed mutagenesis of two conserved cysteine residues (Cys36 and Cys46) in the mature MC69 impaired function of MC69 without affecting its secretion, suggesting the importance of the disulfide bond in MC69 pathogenicity function. Furthermore, deletion of the MC69 orthologous gene reduced pathogenicity of the cucumber anthracnose fungus Colletotrichum orbiculare on both cucumber and Nicotiana benthamiana leaves. We conclude that MC69 is a secreted pathogenicity protein commonly required for infection of two different plant pathogenic fungi, M. oryzae and C. orbiculare pathogenic on monocot and dicot plants, respectively.

Characterization of endo-1,3-1,4-ß-glucanases in GH family 12 from Magnaporthe oryzae.

We have cloned three putative endoglucanase cDNAs, designated MoCel12A, MoCel12B, and MoCel12C, from Magnaporthe oryzae. The deduced peptide sequences of both MoCel12A and MoCel12B contain secretion signal peptides and a catalytic core domain that classify them into GH subfamily 12-1. In contrast, the deduced peptide sequence of MoCel12C consists of a signal peptide, a catalytic core domain, and a fungal-type carbohydrate binding module belonging to GH subfamily 12-2. Although most GH family 12 endoglucanases hydrolyze ß-1,4-glucans such as carboxymethylcellulose or phosphoric acid-swollen cellulose, MoCel12A that was prepared by overexpression in M. oryzae and Brevibacillus choshinensis hydrolyzed specifically 1,3-1,4-ß-glucans, such as barley ß-glucan and lichenan. The specific activity of MoCel12A overexpressed in M. oryzae was about 20 times higher than that prepared from B. choshinensis. Furthermore, MoCel12B prepared by overexpression in B. choshinensis also revealed preferential hydrolysis of endo-1,3-1,4-ß-glucans with limited hydrolysis on carboxymethylcellulose. In comparison with MoCel12A, the activity of MoCel12B was more stable under alkaline conditions. Levels of mRNA encoding MoCel12A were constitutively high during infection and spore formation. The overexpression and disruption of the MoCel12A gene did not affect germination, appressorium formation, or invasion rate; however, M. oryzae overexpressing MoCel12A produced larger numbers of spores than the wild type or a mutant in which the MoCel12A gene was disrupted. These results suggest that MoCel12A functions in part to hydrolyze 1,3-1,4-ß-glucan during infection and spore formation.

Metabolome analysis of response to oxidative stress in rice suspension cells overexpressing cell death suppressor Bax inhibitor-1.

Bax inhibitor-1 (BI-1) is a cell death suppression factor widely conserved in higher plants and animals. Overexpression of Arabidopsis BI-1 (AtBI-1) in plants confers tolerance to various cell death-inducible stresses. However, apart from the cell death-suppressing activity, little is known about the physiological roles of BI-1-overexpressing plants. In this study, we evaluated the effects of AtBI-1 overexpression on the rice metabolome in response to oxidative stress. AtBI-1-overexpressing rice cells in suspension displayed enhanced tolerance to menadione-induced oxidative stress compared with vector control cells, whereas AtBI-1 overexpression did not influence the increase of intracellular H(2)O(2) concentration or inhibition of oxidative stress-sensitive aconitase activity. Capillary electrophoresis-mass spectrometry (CE-MS)-based metabolome analysis revealed dynamic metabolic changes in oxidatively stressed rice cells, e.g. depletion of the central metabolic pathway, imbalance of the redox state and energy charge, and accumulation of amino acids. Furthermore, comparative metabolome analysis demonstrated that AtBI-1 overexpression did not affect primary metabolism in rice cells under normal growth conditions but significantly altered metabolite composition within several distinct pathways under cell death-inducible oxidative stress. The AtBI-1-mediated metabolic alteration included recovery of the redox state and energy charge, which are known as important factors for metabolic defense against oxidative stress. These observations suggest that although AtBI-1 does not affect rice metabolism directly, its cell death suppression activity leads to enhanced capacity to acclimate oxidative stress.

SPM1 encoding a vacuole-localized protease is required for infection-related autophagy of the rice blast fungus Magnaporthe oryzae.

SPM1, encoding a putative subtilisin-like protease, is involved in pathogenicity of the rice blast fungus Magnaporthe oryzae, but its detailed function remains unknown. Here, we report that SPM1 encodes a vacuole-localized protease that is a critical component for autophagy during the infection process of M. oryzae. Detailed phenotypic analysis of targeted disruption mutants of SPM1 revealed that the mutants have pleiotropic defects in infection-related steps including germination, appressorium formation, host invasion and postinvasive growth, indicating the requirement of Spm1 function for the broad phase of infection. It has been shown that the Spm1 homolog of yeast functions in autophagy, the degradation machinery mediated by vacuoles, implying the involvement of Spm1 in autophagy in M. oryzae. In-gel protease activity assay of the recombinant Spm1 protein indicated that Spm1 had a protease activity. An Spm1-GFP fusion protein was detected inside vacuoles of fungal cells, indicating that Spm1 is a protease localized in vacuoles. Furthermore, degradation of putative autophagic bodies was retarded in vacuoles of the spm1 mutant. These data strongly suggest that SPM1-encoded protease functions in autophagy required for the pathogenicity of M. oryzae.

Association genetics reveals three novel avirulence genes from the rice blast fungal pathogen Magnaporthe oryzae.

To subvert rice (Oryza sativa) host defenses, the devastating ascomycete fungus pathogen Magnaporthe oryzae produces a battery of effector molecules, including some with avirulence (AVR) activity, which are recognized by host resistance (R) proteins resulting in rapid and effective activation of innate immunity. To isolate novel avirulence genes from M. oryzae, we examined DNA polymorphisms of secreted protein genes predicted from the genome sequence of isolate 70-15 and looked for an association with AVR activity. This large-scale study found significantly more presence/absence polymorphisms than nucleotide polymorphisms among 1032 putative secreted protein genes. Nucleotide diversity of M. oryzae among 46 isolates of a worldwide collection was extremely low (theta=8.2x10(-5)), suggestive of recent pathogen dispersal. However, no association between DNA polymorphism and AVR was identified. Therefore, we used genome resequencing of Ina168, an M. oryzae isolate that contains nine AVR genes. Remarkably, a total of 1.68 Mb regions, comprising 316 candidate effector genes, were present in Ina168 but absent in the assembled sequence of isolate 70-15. Association analyses of these 316 genes revealed three novel AVR genes, AVR-Pia, AVR-Pii, and AVR-Pik/km/kp, corresponding to five previously known AVR genes, whose products are recognized inside rice cells possessing the cognate R genes. AVR-Pia and AVR-Pii have evolved by gene gain/loss processes, whereas AVR-Pik/km/kp has evolved by nucleotide substitutions and gene gain/loss.

A high-throughput screen of cell-death-inducing factors in Nicotiana benthamiana identifies a novel MAPKK that mediates INF1-induced cell death signaling and non-host resistance to Pseudomonas cichorii.

A high-throughput overexpression screen of Nicotiana benthamiana cDNAs identified a gene for a mitogen-activated protein kinase kinase (MAPKK) as a potent inducer of the hypersensitive response (HR)-like cell death. NbMKK1 protein is localized to the nucleus, and the N-terminal putative MAPK docking site of NbMKK1 is required for its function as a cell-death inducer. NbMKK1-mediated leaf-cell death was compromised in leaves where NbSIPK expression was silenced by virus-induced gene silencing. A yeast two-hybrid assay showed that NbMKK1 and NbSIPK physically interact, suggesting that NbSIPK is one of the downstream targets of NbMKK1. Phytophthora infestans INF1 elicitor-mediated HR was delayed in NbMKK1-silenced plants, indicating that NbMKK1 is involved in this HR pathway. Furthermore, the resistance of N. benthamiana to a non-host pathogen Pseudomonas cichorii was compromised in NbMKK1-silenced plants. These results demonstrate that MAPK cascades involving NbMKK1 control non-host resistance including HR cell death.

A Novel MAPKK Involved in Cell Death and Defense Signaling.

A high-throughput in planta overexpression screen of a Nicotiana benthamiana cDNA library identified a mitogen activated protein kinase kinase (MAPKK), NbMKK1, as a potent inducer of hypersensitive response (HR)-like cell death. NbMKK1-mediated cell death was attenuated in plants whereby expression of NbSIPK, an ortholog of tobacco SIPK and Arabidopsis AtMPK6, was knocked down by virus-induced gene silencing (VIGS), suggesting that NbMKK1 functions upstream of NbSIPK. In accordance with this result, NbMKK1 phosphorylated NbSIPK in vitro, and furthermore NbMKK1 and NbSIPK physically interacted in yeast two-hybrid assay. VIGS of NbMKK1 in N. benthamiana resulted in a delay of Phytophthora infestans INF1 elicitin-mediated HR as well as in the reduction of resistance against a non-host pathogen Pseudomonas cichorii. Our data of NbMKK1, together with that of LeMKK4,1 demonstrate the presence of a novel defense signaling pathway involving NbMKK1/LeMKK4 and SIPK.

Regulatory mechanisms of ROI generation are affected by rice spl mutations.

Reactive oxygen intermediates (ROIs) play a pivotal role in the hypersensitive response (HR) in disease resistance. NADPH oxidase is a major source of ROI; however, the mechanisms of its regulation are unclear. Rice spl mutants spontaneously form lesions which resemble those occurring during the HR, suggesting that the mutations affect regulation of the HR. We found that spl2, spl7 and spl11 mutant cells accumulated increased amounts of H(2)O(2) in response to rice blast fungal elicitor. Increased accumulation of ROIs was suppressed by inhibition of NADPH oxidase in the spl cells, and was also observed in the ozone-exposed spl plants. These mutants have sufficient activities of ROI-scavenging enzymes compared with the wild type. In addition, spl7 mutant cells accumulated higher amounts of H(2)O(2) when treated with calyculin A (CA), an inhibitor of protein phosphatase. Furthermore, spl2 mutant plants exhibited accelerated accumulation of H(2)O(2) and increased rates of cell death in response to wounding. These results suggest that the spl2, spl7 and spl11 mutants are defective in the regulation of NADPH oxidase, and the spl7 mutation may give rise to enhancement of the signaling pathway which protein dephosphorylation controls, while the spl2 mutation affects both the pathogen-induced and wound-induced signaling pathways.

High-throughput in planta expression screening identifies a class II ethylene-responsive element binding factor-like protein that regulates plant cell death and non-host resistance.

We performed high-throughput screening using the potato virus X (PVX) system to overexpress Nicotiana benthamiana genes in planta and identify positive regulators of cell death. This screening identified NbCD1, a novel class II ethylene-responsive element binding factor (ERF), as a potent inducer of the hypersensitive response (HR)-like cell death. NbCD1 expression was induced by treatments with INF1 elicitor and a non-host pathogen Pseudomonas cichorii. NbCD1 exhibited transcriptional repressor activity through its EAR motif, and this motif was necessary for NbCD1 to cause cell death. We identified 58 genes that displayed altered transcription following NbCD1 overexpression. NbCD1 overexpression downregulated the expression of HSR203, a negative regulator of hypersensitive death. Conditional expression of NbCD1 in Arabidopsis also caused cell death, indicating that NbCD1 downstream cascades are conserved in dicot plants. To further confirm the role of NbCD1 in defense, we used virus-induced gene silencing to demonstrate that NbCD1 is required for non-host resistance of N. benthamiana to the bacterial pathogen P. cichorii. Our data point to a model of transcriptional regulatory cascades. NbCD1 positively regulates cell death and contributes to non-host resistance, possibly by downregulating the expression of other defense response genes.