In Vitro and In Vivo Anti-Inflammatory Effects of Cannabidiol Isolated from Novel Hemp (Cannabis sativa L.) Cultivar Pink Pepper.

Cannabis sativa L. contains more than 80 cannabinoids, among which cannabidiol (CBD) is the main neuroactive component. We aimed to investigate the anti-inflammatory efficacy of CBD in vitro and in vivo isolated from "Pink pepper", a novel hemp cultivar, by repeating the method of selecting and cultivating individuals with the highest CBD content. We investigated the effects of CBD on inflammatory markers elevated by lipopolysaccharide (LPS) treatment in RAW 264.7 mouse macrophage cells through Western blot and RT-PCR. In addition, we confirmed these effects through the ELISA of inflamed paw tissue of a λ-carrageenan-induced mouse edema model that received an oral administration of CBD. CBD inhibited the LPS-induced phosphorylation of NF-κB and MAPK in RAW 264.7 and exhibited anti-inflammatory effects by participating in these pathways. In our in vivo study, we confirmed that CBD also inhibited the inflammatory mediators of proteins extracted from edematous mouse paw tissue. These results show that CBD isolated from "Pink pepper" exhibits potent anti-inflammatory effects. These anti-inflammatory effects of CBD have pharmacological and physiological significance, highlighting the industrial value of this novel cultivar.

The human PMR1 endonuclease stimulates cell motility by down regulating miR-200 family microRNAs.

The motility of MCF-7 cells increases following expression of a human PMR1 transgene and the current study sought to identify the molecular basis for this phenotypic change. Ensemble and single cell analyses show increased motility is dependent on the endonuclease activity of hPMR1, and cells expressing active but not inactive hPMR1 invade extracellular matrix. Nanostring profiling identified 14 microRNAs that are downregulated by hPMR1, including all five members of the miR-200 family and others that also regulate invasive growth. miR-200 levels increase following hPMR1 knockdown, and changes in miR-200 family microRNAs were matched by corresponding changes in miR-200 targets and reporter expression. PMR1 preferentially cleaves between UG dinucleotides within a consensus YUGR element when present in the unpaired loop of a stem-loop structure. This motif is present in the apical loop of precursors to most of the downregulated microRNAs, and hPMR1 targeting of pre-miRs was confirmed by their loss following induced expression and increase following hPMR1 knockdown. Introduction of miR-200c into hPMR1-expressing cells reduced motility and miR-200 target gene expression, confirming hPMR1 acts upstream of Dicer processing. These findings identify a new role for hPMR1 in the post-transcriptional regulation of microRNAs in breast cancer cells.

SR proteins in vertical integration of gene expression from transcription to RNA processing to translation.

SR proteins have been studied extensively as a family of RNA-binding proteins that participate in both constitutive and regulated pre-mRNA splicing in mammalian cells. However, SR proteins were first discovered as factors that interact with transcriptionally active chromatin. Recent studies have now uncovered properties that connect these once apparently disparate functions, showing that a subset of SR proteins seem to bind directly to the histone 3 tail, play an active role in transcriptional elongation, and colocalize with genes that are engaged in specific intra- and interchromosome interactions for coordinated regulation of gene expression in the nucleus. These transcription-related activities are also coupled with a further expansion of putative functions of specific SR protein family members in RNA metabolism downstream of mRNA splicing, from RNA export to stability control to translation. These findings, therefore, highlight the broader roles of SR proteins in vertical integration of gene expression and provide mechanistic insights into their contributions to genome stability and proper cell-cycle progression in higher eukaryotic cells.

Role of the MoYAK1 protein kinase gene in Magnaporthe oryzae development and pathogenicity.

Conidiation and appressorium differentiation are key processes for polycyclic dissemination and infection in many pathogens. Our previous study using DNA microarray led to the discovery of the MoYAK1 gene in Magnaporthe oryzae that is orthologous to YAK1 in Saccharomyces cerevisiae. Although the mechanistic roles of YAK1 in S. cerevisiae have been described, roles of MoYAK1 in M. oryzae, a phytopathogenic fungus responsible for rice blast, remain uncharacterized. Targeted disruption of MoYAK1 results in pleiotropic defects in M. oryzae development and pathogenicity. The ΔMoyak1 mutant exhibits a severe reduction in aerial hyphal formation and conidiation. Conidia in the ΔMoyak1 are delayed in germination and demonstrate decreased glycogen content in a conidial age-dependent manner. The expression of hydrophobin-coding genes is dramatically changed in the ΔMoyak1 mutant, leading to a loss of surface hydrophobicity. Unlike the complete inability of the ΔMoyak1 mutant to develop appressoria on an inductive surface, the mutant forms appressoria of abnormal morphology in response to exogenous cyclic adenosine-5'-monophosphate and host-driven signals, which are all defective in penetrating host tissues due to abnormalities in glycogen and lipid metabolism, turgor generation and cell wall integrity. These data indicate that MoYAK1 is a protein kinase important for the development and pathogenicity of M. oryzae.

Comparative functional analysis of the velvet gene family reveals unique roles in fungal development and pathogenicity in Magnaporthe oryzae.

The ascomycete fungus Magnaporthe oryzae is an economically important pathogen that causes rice blast disease worldwide. Accumulating evidence indicates that the fungal velvet genes are key regulators of a number of cellular processes, including development, pathogenicity and secondary metabolism, in many species of fungi. In this study, we identified and functionally characterized four genes (MoVOSA, MoVELB, MoVEA, and MoVELC) from the genome of the fungal pathogen M. oryzae. These genes were homologous to the velvet gene family of Aspergillus nidulans. Deletions of MoVEA, MoVELB, and MoVELC resulted in a significant decrease in conidiation, indicating their roles as positive regulators thereof. The MoVELC gene was involved in development of conidial morphology, while MoVELB and MoVEA appeared necessary for conidial germination, MoVEA further being indispensable for appressorial development and modulation of reactive oxygen species in disease development. Deletion of MoVELC affected the cell wall integrity of appressoria, resulting in failure to penetrate host cells. Unexpectedly, MoVOSA appeared dispensable for the development and pathogenicity of M. oryzae, even though its homologs play specific roles in other fungal species. Taken together, our data demonstrate that the velvet genes are linked to M. oryzae infection-related development and pathogenicity, and the findings provide a framework for comparative studies of the conserved velvet gene family across a range of fungal taxa, which may provide new insight into fungal development and pathogenicity.

Identification of the human PMR1 mRNA endonuclease as an alternatively processed product of the gene for peroxidasin-like protein.

The PMR1 endonuclease was discovered in Xenopus liver and identified as a member of the large and diverse peroxidase gene family. The peroxidase genes arose from multiple duplication and rearrangement events, and their high degree of sequence similarity confounded attempts to identify human PMR1. The functioning of PMR1 in mRNA decay depends on the phosphorylation of a tyrosine in the C-terminal polysome targeting domain by c-Src. The sequences of regions that are required for c-Src binding and phosphorylation of Xenopus PMR1 were used to inform a bioinformatics search that identified two related genes as potential candidates for human PMR1: peroxidasin homolog (PXDN) and peroxidasin homolog-like (PXDNL) protein. Although each of these genes is predicted to encode a large, multidomain membrane-bound peroxidase, alternative splicing of PXDNL pre-mRNA yields a transcript whose predicted product is a 57-kDa protein with 42% sequence identity to Xenopus PMR1. Results presented here confirm the existence of the predicted 57-kDa protein, show this is the only form of PXDNL detected in any of the human cell lines examined, and confirm its identity as human PMR1. Like the Xenopus protein, human PMR1 binds to c-Src, is tyrosine phosphorylated, sediments on polysomes, and catalyzes the selective decay of a PMR1 substrate mRNA. Importantly, the expression of human PMR1 stimulates cell motility in a manner similar to that of the Xenopus PMR1 expressed in human cells, thus providing definitive evidence linking endonuclease decay to the regulation of cell motility.

Degeneration of oil bodies by rough endoplasmic reticulum -associated protein during seed germination in Cannabis sativa.

Oil bodies serve as a vital energy source of embryos during germination and contribute to sustaining the initial growth of seedlings until photosynthesis initiation. Despite high stability in chemical properties, how oil bodies break down and go into the degradation process during germination is still unknown. This study provides a morphological understanding of the mobilization of stored compounds in the seed germination of Cannabis. The achenes of fibrous hemp cultivar (Cannabis sativa cv. 'Chungsam') were examined in this study using light microscopy, scanning electron microscopy and transmission electron microscopy. Oil bodies in Cannabis seeds appeared spherical and sporadically distributed in the cotyledonary cells. Protein bodies contained electron-dense globoid and heterogeneous protein matrices. During seed germination, rough endoplasmic reticulum (rER) and high electron-dense substances were present adjacent to the oil bodies. The border of the oil bodies became a dense cluster region and appeared as a sinuous outline. Later, irregular hyaline areas were distributed throughout oil bodies, showing the destabilized emulsification of oil bodies. Finally, the oil bodies lost their morphology and fused with each other. The storage proteins were concentrated in the centre of the protein body as a dense homogenous circular mass surrounded by a light heterogeneous area. Some storage proteins are considered emulsifying agents on the surface region of oil bodies, enabling them to remain stable and distinct within and outside cotyledon cells. At the early germination stage, rER appeared and dense substances aggregated adjacent to the oil bodies. Certain proteins were synthesized within the rER and then translocated into the oil bodies by crossing the half membrane of oil bodies. Our data suggest that rER-associated proteins function as enzymes to lyse the emulsifying proteins, thereby weakening the emulsifying agent on the surface of the oil bodies. This process plays a key role in the degeneration of oil bodies and induces coalescence during seed germination.

In Vitro and In Vivo Anti-Inflammatory Potential of Cannabichromene Isolated from Hemp.

Cannabichromene (CBC), a non-psychoactive cannabinoid found in Cannabis sativa, has recently been shown to possess several medicinal properties. However, how CBC produces anti-inflammatory effects and the mechanisms of this remain poorly studied. Therefore, we extracted and purified the CBC from the Cannabis sativa cv. pink pepper (hemp cultivar). The efficacy of CBC in reducing inflammation in RAW 264.7 macrophages and a λ-carrageenan-induced mouse model was then evaluated. CBC had no cytotoxicity up to a concentration of 20 µM and inhibited nitric oxide production by approximately 50% at a concentration of 20 µM. In addition, CBC treatment significantly inhibited causes of inflammation such as inducible nitric oxide synthase (iNOS), interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor-α (TNF-α) at both the mRNA and protein levels. Moreover, CBC suppressed LPS-stimulated inflammation in RAW 264.7 cells by downregulating the nuclear factor kappa B (NF-kB) and mitogen-activated protein kinase pathways (MAPK). Furthermore, our in vivo experiments confirmed that the λ-carrageenan-induced increase in the levels of the cytokines iNOS, IL-1ß, and IL-6 was abrogated following treatment with CBC. Therefore, CBC has potential anti-inflammatory effects and may be useful for preventing or treating inflammation.

Ultrasonic-Assisted Extraction of Cannabidiolic Acid from Cannabis Biomass.

Industrial hemp (Cannabis spp.) has many compounds of interest with potential medical benefits. Of these compounds, cannabinoids have come to the center of attention, specifically acidic cannabinoids. The focus is turning toward acidic cannabinoids due to their lack of psychotropic activity. Cannabis plants produce acidic cannabinoids with hemp plants producing low levels of psychotropic cannabinoids. As such, utilization of hemp for acidic cannabinoid extraction would eliminate the need for decarboxylation prior to extraction as a source for the cannabinoids. The use of solvent-based extraction is ideal for obtaining acidic cannabinoids as their solubility in solvents such as supercritical CO2 is limited due to the high pressure and temperature required to reach their solubility constants. An alternative method designed to increase solubility is ultrasonic-assisted extraction. In this protocol, the impact of solvent polarity (acetonitrile 0.46, ethanol 0.65, methanol 0.76, and water 1.00) and concentration (20%, 50%, 70%, 90%, and 100%) on ultrasonic-assisted extraction efficiency has been examined. Results show that water was the least effective and acetonitrile was the most effective solvent examined. Ethanol was further examined since it has the lowest toxicity and is generally regarded as safe (GRAS). Surprisingly, 50% ethanol in water is the most effective ethanol concentration for extracting the highest amount of cannabinoids from hemp. The increase in cannabidiolic acid concentration was 28% when compared to 100% ethanol, and 23% when compared to 100% acetonitrile. While it was determined that 50% ethanol is the most effective concentration for our application, the method has also been demonstrated to be effective with alternative solvents. Consequently, the proposed method is deemed effective and rapid for extracting acidic cannabinoids.

Antioxidant and lipid-reducing effects of Rosa rugosa root extract in 3T3-L1 cell.

Rosa rugosa root is traditionally known to be effective in the treatment of diabetes in Korea. R. rugosa root-specific compounds also show antioxidant effects, and could reduce lipid and fat accumulation, however, the underlying mechanism has not been clarified. In present study, the antioxidant and lipid-reducing effects of a 50% ethanol extract of R. rugosa (REE) were investigated differentiated mouse preadipocytes (3T3-L1 cells). REE showed strong radical scavenging activities and inhibitory effect of total lipid accumulation and triglyceride levels in differentiated 3T3-L1 cells. In addition, REE treatment reduced the mRNA and protein levels of adipogenesis and lipogenesis markers. This REE-promoted lipid reduction was caused by downregulation of peroxisome proliferator activated receptor gamma (PPARγ), CCAAT/enhancer binding protein alpha (C/EBPα), and sterol regulatory element binding protein1 (SREBP1c) and down regulation of ERK expression. Overall, these results demonstrate the potential of REE for development of a drug in the medical treatment of lipid-associated disorders.

Employing Aeroponic Systems for the Clonal Propagation of Cannabis.

This protocol describes the standardization of an efficient clonal propagation technique of hemp by utilizing aeroponic systems. Primary shoot cuttings were excised from two hemp varieties, named "Cherry Wine" and "Red Robin" (17-20% w/w CBD), that served as 'mother plant'. An auxin precursor (indole-3-butyric acid) was applied to stimulate root development in the basal portion of the excised cuttings prior to placement in the system. Cuttings were lightly misted with the nutrient mist solution every three days to provide nutritional support as the solution contains the essential macronutrients, including nitrogen, phosphorus, and potassium. The aeroponic system water reservoir maintained a pH range between 5.0-6.0 and a water temperature between 20-22 °C. A submersible water pump was used to deliver water to the cuttings. The shoot tip cuttings were provided with 24 h of light per day for 10 days until root development occurred, upon which the rooted cuttings were transplanted for research purposes. These aeroponic systems have proven to generate desirable results for Cannabis propagation. The method described here alleviates potential time constraints that arise from traditional methods to allow for a more efficient means for the asexual propagation of Cannabis.

Tobacco Hornworm as an Insect Model System for Cannabinoid Pre-clinical Studies.

With increased attention on cannabinoids in medicine, several mammalian model organisms have been used to elucidate their unknown pharmaceutical functions. However, many difficulties remain in mammalian research, which necessitates the development of non-mammalian model organisms for cannabinoid research. The authors suggest the tobacco hornworm Manduca sexta as a novel insect model system. This protocol provides information on preparing the artificial diet with varying amounts of cannabidiol (CBD), setting up a cultivation environment, and monitoring their physiological and behavioral changes in response to CBD treatment. Briefly, upon receiving hornworm eggs, the eggs were allowed 1-3 days at 25 °C on a 12:12 light-dark cycle to hatch before being randomly distributed into control (wheat germ-based artificial diet; AD), vehicle (AD + 0.1% medium-chain triglyceride oil; MCT oil) and treatment groups (AD + 0.1% MCT + 1 mM or 2 mM of CBD). Once the media was prepared, 1st instar larvae were individually placed in a 50 mL test tube with a wooden skewer stick, and then the test tube was covered with a cheesecloth. Measurements were taken in 2-day intervals for physiological and behavioral responses to the CBD administration. This simple cultivation procedure allows researchers to test large specimens in a given experiment. Additionally, the relatively short life cycles enable researchers to study the impact of cannabinoid treatments over multiple generations of a homogenous population, allowing for data to support an experimental design in higher mammalian model organisms.

The Membrane-Bound Protein, MoAfo1, Is Involved in Sensing Diverse Signals from Different Surfaces in the Rice Blast Fungus.

To establish an infection, fungal pathogens must recognize diverse signals from host surfaces. The rice blast fungus, Magnaporthe oryzae, is one of the best models studying host-pathogen interactions. This fungus recognizes physical or chemical signals from the host surfaces and initiates the development of an infection structure called appressorium. Here, we found that protein MoAfo1(appressorium formation, MGG_10422) was involved in sensing signal molecules such as cutin monomers and long chain primary alcohols required for appressorium formation. The knockout mutant (ΔMoafo1) formed a few abnormal appressoria on the onion and rice sheath surfaces. However, it produced normal appressoria on the surface of rice leaves. MoAfo1 localized to the membranes of the cytoplasm and vacuole-like organelles in conidia and appressoria. Additionally, the ΔMoafo1 mutant showed defects in appressorium morphology, appressorium penetration, invasive growth, and pathogenicity. These multiple defects might be partially due to failure to respond properly to oxidative stress. These findings broaden our understanding of the fungal mechanisms at play in the recognition of the host surface during rice blast infection.

Homeobox Transcription Factors Are Required for Fungal Development and the Suppression of Host Defense Mechanisms in the Colletotrichum scovillei-Pepper Pathosystem.

Colletotrichum scovillei, an ascomycete phytopathogenic fungus, is the main causal agent of serious yield losses of economic crops worldwide. The fungus causes anthracnose disease on several fruits, including peppers. However, little is known regarding the underlying molecular mechanisms involved in the development of anthracnose caused by this fungus. In an initial step toward understanding the development of anthracnose on pepper fruits, we retrieved 624 transcription factors (TFs) from the whole genome of C. scovillei and comparatively analyzed the entire repertoire of TFs among phytopathogenic fungi. Evolution and proliferation of members of the homeobox-like superfamily, including homeobox (HOX) TFs that regulate the development of eukaryotic organisms, were demonstrated in the genus Colletotrichum. C. scovillei was found to contain 10 HOX TF genes (CsHOX1 to CsHOX10), which were functionally characterized using deletion mutants of each CsHOX gene. Notably, CsHOX1 was identified as a pathogenicity factor required for the suppression of host defense mechanisms, which represents a new role for HOX TFs in pathogenic fungi. CsHOX2 and CsHOX7 were found to play essential roles in conidiation and appressorium development, respectively, in a stage-specific manner in C. scovillei. Our study provides a molecular basis for understanding the mechanisms associated with the development of anthracnose on fruits caused by C. scovillei, which will aid in the development of novel approaches for disease management. IMPORTANCE The ascomycete phytopathogenic fungus, Colletotrichum scovillei, causes serious yield loss on peppers. However, little is known about molecular mechanisms involved in the development of anthracnose caused by this fungus. We analyzed whole-genome sequences of C. scovillei and isolated 624 putative TFs, revealing the existence of 10 homeobox (HOX) transcription factor (TF) genes. We found that CsHOX1 is a pathogenicity factor required for the suppression of host defense mechanism, which represents a new role for HOX TFs in pathogenic fungi. We also found that CsHOX2 and CsHOX7 play essential roles in conidiation and appressorium development, respectively, in a stage-specific manner in C. scovillei. Our study contributes to understanding the mechanisms associated with the development of anthracnose on fruits caused by C. scovillei, which will aid for initiating novel approaches for disease management.

Antioxidant and Anti-Obesity Activities of Polygonum cuspidatum Extract through Alleviation of Lipid Accumulation on 3T3-L1 Adipocytes.

Natural products are widely used due to their various biological activities which include antiinflammatory, antioxidant, and anti-obesity effects. In this study, we determined the antioxidative and anti-obesity effects of Polygonum cuspidatum 50% ethanol extract (PEE). The antioxidative effect of PEE was evaluated using its radical scavenging activity, total phenolic content, and reducing power. The anti-obesity effect of PEE was investigated using 3T3-L1 adipocytes. The antioxidative activity of PEE was progressively increased in various concentrations, mainly due to the presence of phenolic compounds. PEE also alleviated lipid accumulation on 3T3-L1 adipocytes and downregulated the mRNA and protein production of adipogenesis-related (SREBP-1c, PPARγ, C/EBPα) and lipogenesis-related (aP2, FAS, ACC) markers. Furthermore, we found that the inhibitory effect on lipid accumulation via PEE was caused by the alleviation of NF-κB, p38 MAPK, ERK1/2, and JNK at the protein level. Taken together, our results imply that PEE is a potential antioxidant that can prevent obesityassociated disorders.

Optimization of Polyethylene Glycol-Mediated Transformation of the Pepper Anthracnose Pathogen Colletotrichum scovillei to Develop an Applied Genomics Approach.

Colletotrichum acutatum is a species complex responsible for anthracnose disease in a wide range of host plants. Strain C. acutatum KC05, which was previously isolated from an infected pepper in Gangwon Province of South Korea, was reidentified as C. scovillei using combined sequence analyses of multiple genes. As a prerequisite for understanding the pathogenic development of the pepper anthracnose pathogen, we optimized the transformation system of C. scovillei KC05. Protoplast generation from young hyphae of KC05 was optimal in an enzymatic digestion using a combined treatment of 2% lysing enzyme and 0.8% driselase in 1 M NH4Cl for 3 h incubation. Prolonged incubation for more than 3 h decreased protoplast yields. Protoplast growth of KC05 was completely inhibited for 4 days on regeneration media containing 200 µg/ml hygromycin B, indicating the viability of this antibiotic as a selection marker. To evaluate transformation efficiency, we tested polyethylene glycol-mediated protoplast transformation of KC05 using 19 different loci found throughout 10 (of 27) scaffolds, covering approximately 84.1% of the entire genome. PCR screening showed that the average transformation efficiency was about 17.1% per 100 colonies. Southern blot analyses revealed that at least one transformant per locus had single copy integration of PCR-screened positive transformants. Our results provide valuable information for a functional genomics approach to the pepper anthracnose pathogen C. scovillei.

A New Record and Characterization of Asparagus Purple Spot Caused by Stemphylium vesicarium in Korea.

In 2017, small, elliptical, brownish purple spots on spears and ferns of asparagus were found in fields of Gangwon-do. The isolated fungal species was identified as an ascomycete Stemphylium vesicarium based on morphological characteristics and molecular phylogenic analyses including nucleotide sequences of the internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and cytochrome b (cytb). A pathogenicity test revealed that S. vesicarium was the causal agent of purple spot disease on asparagus. The occurrence of purple spots caused by S. vesicarium on asparagus is the first report in Korea.

A PAS-Containing Histidine Kinase is Required for Conidiation, Appressorium Formation, and Disease Development in the Rice Blast Fungus, Magnaporthe oryzae.

Rice blast disease, caused by the ascomycete fungus Magnaporthe oryzae, is one of the most important diseases in rice production. PAS (period circadian protein, aryl hydrocarbon receptor nuclear translocator protein, single-minded protein) domains are known to be involved in signal transduction pathways, but their functional roles have not been well studied in fungi. In this study, targeted gene deletion was carried out to investigate the functional roles of the PAS-containing gene MoPAS1 (MGG_02665) in M. oryzae. The deletion mutant ΔMopas1 exhibited easily wettable mycelia, reduced conidiation, and defects in appressorium formation and disease development compared to the wild type and complemented transformant. Exogenous cAMP restored appressorium formation in ΔMopas1, but the shape of the restored appressorium was irregular, indicating that MoPAS1 is involved in sensing the hydrophobic surface. To examine the expression and localization of MoPAS1 in M. oryzae during appressorium development and plant infection, we constructed a MoPAS1:GFP fusion construct. MoPAS1:GFP was observed in conidia and germ tubes at 0 and 2 h post-infection (hpi) on hydrophobic cover slips. By 8 hpi, most of the GFP signal was observed in the appressoria. During invasive growth in host cells, MoPAS1:GFP was found to be fully expressed in not only the appressoria but also invasive hyphae, suggesting that MoPAS may contribute to disease development in host cells. These results expand our knowledge of the roles of PAS-containing regulatory genes in the plant-pathogenic fungus M. oryzae.

Distinct roles of the YPEL gene family in development and pathogenicity in the ascomycete fungus Magnaporthe oryzae.

Members of the Yippee-like (YPEL) gene family are highly conserved in eukaryotes and are homologous to the Drosophila yippee gene. In this study, we functionally characterized two YPEL-homologous genes, MoYPEL1 and MoYPEL2, in the rice blast pathogen Magnaporthe oryzae using the deletion mutants ΔMoypel1, ΔMoypel2, and ΔΔMoypel1,2. The MoYPEL1 deletion mutant was significantly defective in conidiation and unable to undergo appressorium development; however, deletion of MoYPEL2 resulted in a significant increase in conidiation and the abnormal development of two appressoria per conidium. These data demonstrate the opposite roles of each member of the YPEL gene family during the development of M. oryzae. The double mutant was phenotypically similar to the ΔMoypel1 mutant in conidiation, but similar to the ΔMoypel2 mutant in appressorium development. Subcellular localization of the MoYPEL1 protein was dynamic during appressorium development, while the MoYPEL2 protein consistently localized within the nuclei during developmental stages. Our studies indicate that the two YPEL gene family members play distinct roles in the developmental stages of M. oryzae, furthering our understanding of disease dissemination and development in fungi.

A Small GTPase RHO2 Plays an Important Role in Pre-infection Development in the Rice Blast Pathogen Magnaporthe oryzae.

The rice blast pathogen Magnaporthe oryzae is a global threat to rice production. Here we characterized RHO2 gene (MGG_02457) that belongs to the Rho GTPase family, using a deletion mutant. This mutant ΔMorho2 exhibited no defects in conidiation and germination but developed only 6% of appressoria in response to a hydrophobic surface when compared to the wild-type progenitor. This result indicates that MoRHO2 plays a role in appressorium development. Furthermore, exogenous cAMP treatment on the mutant led to appressoria that exhibited abnormal morphology on both hydrophobic and hydrophilic surfaces. These outcomes suggested the involvement of MoRHO2 in cAMP-mediated appressorium development. ΔMorho2 mutation also delayed the development of appressorium-like structures (ALS) at hyphal tips on hydrophobic surface, which were also abnormally shaped. These results suggested that MoRHO2 is involved in morphological development of appressoria and ALS from conidia and hyphae, respectively. As expected, ΔMorho2 mutant was defective in plant penetration, but was still able to cause lesions, albeit at a reduced rate on wounded plants. These results implied that MoRHO2 plays a role in M. oryzae virulence as well.