Zinc deficiency-induced defensin-like proteins are involved in the inhibition of root growth in Arabidopsis.

The depletion of cellular zinc (Zn) adversely affects plant growth. Plants have adaptation mechanisms for Zn-deficient conditions, inhibiting growth through the action of transcription factors and metal transporters. We previously identified three defensin-like (DEFL) proteins (DEFL203, DEFL206 and DEFL208) that were induced in Arabidopsis thaliana roots under Zn-depleted conditions. DEFLs are small cysteine-rich peptides involved in defense responses, development and excess metal stress in plants. However, the functions of DEFLs in the Zn-deficiency response are largely unknown. Here, phylogenetic tree analysis revealed that seven DEFLs (DEFL202-DEFL208) were categorized into one subgroup. Among the seven DEFLs, the transcripts of five (not DEFL204 and DEFL205) were upregulated by Zn deficiency, consistent with the presence of cis-elements for basic-region leucine-zipper 19 (bZIP19) or bZIP23 in their promoter regions. Microscopic observation of GFP-tagged DEFL203 showed that DEFL203-sGFP was localized to the apoplast and plasma membrane. Whereas a single mutation of the DEFL202 or DEFL203 genes only slightly affected root growth, defl202 defl203 double mutants showed enhanced root growth under all growth conditions. We also showed that the size of the root meristem was increased in the double mutants compared with the wild type. Our results suggest that DEFL202 and DEFL203 are redundantly involved in the inhibition of root growth under Zn-deficient conditions through a reduction in root meristem length and cell number.

Arabidopsis thaliana Subclass I ACTIN DEPOLYMERIZING FACTORs Regulate Nuclear Organization and Gene Expression.

ACTIN DEPOLYMERIZING FACTOR (ADF) is a conserved protein that regulates the organization and dynamics of actin microfilaments. Eleven ADFs in the Arabidopsis thaliana genome are grouped into four subclasses, and subclass I ADFs, ADF1-4, are all expressed throughout the plant. Previously, we showed that subclass I ADFs function in the regulation of the response against powdery mildew fungus as well as in the regulation of cell size and endoreplication. Here, we report a new role of subclass I ADFs in the regulation of nuclear organization and gene expression. Through microscopic observation of epidermal cells in mature leaves, we found that the size of chromocenters in both adf4 and transgenic lines where expression of subclass I ADFs is downregulated (ADF1-4Ri) was reduced compared with that of wild-type Col-0. Arabidopsis thaliana possesses eight ACTIN (ACT) genes, among which ACT2, -7 and -8 are expressed in vegetative organs. The chromocenter size in act7, but not in the act2/8 double mutant, was enlarged compared with that in Col-0. Microarray analysis revealed that 1,818 genes were differentially expressed in adf4 and ADF1-4Ri. In particular, expression of 22 nucleotide-binding leucine-rich repeat genes, which are involved in effector-triggered plant immunity, was reduced in adf4 and ADF1-4Ri. qRT-PCR confirmed the altered expressions shown with microarray analysis. Overall, these results suggest that ADF regulates various aspects of plant physiology through its role in regulation of nuclear organization and gene expression. The mechanism how ADF and ACT regulate nuclear organization and gene expression is discussed.

Role of gut microbiota in sex- and diet-dependent metabolic disorders that lead to early mortality of androgen receptor-deficient male mice.

The gut microbiota is involved in metabolic disorders induced by androgen deficiency after sexual maturation in males (late-onset hypogonadism). However, its role in the energy metabolism of congenital androgen deficiency (e.g., androgen-insensitive syndrome) remains elusive. Here, we examined the link between the gut microbiota and metabolic disease symptoms in androgen receptor knockout (ARKO) mouse by administering high-fat diet (HFD) and/or antibiotics. HFD-fed male, but not standard diet-fed male or HFD-fed female, ARKO mice exhibited increased feed efficiency, obesity with increased visceral adipocyte mass and hypertrophy, hepatic steatosis, glucose intolerance, insulin resistance, and loss of thigh muscle. In contrast, subcutaneous fat mass accumulated in ARKO mice irrespective of the diet and sex. Notably, all HFD-dependent metabolic disorders observed in ARKO males were abolished after antibiotics administration. The ratios of fecal weight-to-food weight and cecum weight-to-body weight were specifically reduced by ARKO in HFD-fed males. 16S rRNA sequencing of fecal microbiota from HFD-fed male mice revealed differences in microbiota composition between control and ARKO mice. Several genera or species (e.g., Turicibacter and Lactobacillus reuteri, respectively) were enriched in ARKO mice, and antibiotics treatment spoiled the changes. Furthermore, the life span of HFD-fed ARKO males was shorter than that of control mice, indicating that androgen deficiency causes metabolic dysfunctions leading to early death. These findings also suggest that AR signaling plays a role in the prevention of metabolic dysfunctions, presumably by influencing the gut microbiome, and improve our understanding of health consequences in subjects with hypogonadism and androgen insensitivity.

Gcorn Plant: A Database for Retrieving Functional and Evolutionary Traits of Plant Genes.

Gene homology helps us understand gene function and speciation. The number of plant genes and species registered in public databanks is continuously increasing. It is useful to associate homologous genes of various plants to better understand plant speciation. We designed the Gcorn plant database for the retrieval of information on homology and evolution of a plant gene of interest. Amino acid sequences of 73 species (62 land plants and 11 green algae), containing 2,682,261 sequences, were obtained from the National Center for Biotechnology Information (NCBI) Reference Sequence database. Based on NCBI BLAST searches between these sequences, homologous genes were grouped at various thresholds of homology indices devised by the authors. To show functional and evolutionary traits of a gene of interest, a phylogenetic tree, connecting genes with high homology indices, and line charts of the numbers of genes with various homology indices, are depicted. In addition, such indices are projected on a network graph in which species studied are connected based on the ratios of homologous genes, and on a phylogenetic tree for species based on NCBI Taxonomy. Gcorn plant provides information on homologous genes at various virtual time points along with speciation in plants.

Identification of putative target genes of bZIP19, a transcription factor essential for Arabidopsis adaptation to Zn deficiency in roots.

Zinc (Zn) depletion adversely affects plant growth. To avoid lethal depletion of cellular Zn, plants have evolved mechanisms to adjust the expression of genes associated with Zn homeostasis, the details of which are poorly understood. In the present study, we isolated an Arabidopsis thaliana T-DNA insertion mutant that exhibited hypersensitivity to Zn depletion. By monitoring root development under Zn-deficient conditions, we isolated a single mutant lacking the basic-region leucine-zipper transcription factor gene bZIP19. To identify proteins whose expression is affected by bZIP19, an iTRAQ-based quantitative proteomics analysis was performed using microsomal proteins from wild-type and the bzip19 mutant A. thaliana roots grown on Basal and Zn-deficient media. Of the 797 proteins identified, expression of two members of the Zrt- and Irt-related protein family, ZIP3 and ZIP9, and three defensin-like family proteins was markedly induced in wild-type but not in the bzip19 mutant under Zn-deficient conditions. Furthermore, selected reaction monitoring and quantitative real-time PCR revealed that ZIP9 expression is mediated by bZIP19 and may be partly supported by bZIP23, a homolog of bZIP19. Mutant analysis revealed that ZIP9 is involved in uptake of Zn by the roots, and the mutant lacking ZIP9 was significantly more sensitive to Zn depletion than the wild-type. These results demonstrate that bZIP19 mainly contributes to expression of genes, such as ZIP9, under Zn-deficient conditions.

Multiomics in grape berry skin revealed specific induction of the stilbene synthetic pathway by ultraviolet-C irradiation.

Grape (Vitis vinifera) accumulates various polyphenolic compounds, which protect against environmental stresses, including ultraviolet-C (UV-C) light and pathogens. In this study, we looked at the transcriptome and metabolome in grape berry skin after UV-C irradiation, which demonstrated the effectiveness of omics approaches to clarify important traits of grape. We performed transcriptome analysis using a genome-wide microarray, which revealed 238 genes up-regulated more than 5-fold by UV-C light. Enrichment analysis of Gene Ontology terms showed that genes encoding stilbene synthase, a key enzyme for resveratrol synthesis, were enriched in the up-regulated genes. We performed metabolome analysis using liquid chromatography-quadrupole time-of-flight mass spectrometry, and 2,012 metabolite peaks, including unidentified peaks, were detected. Principal component analysis using the peaks showed that only one metabolite peak, identified as resveratrol, was highly induced by UV-C light. We updated the metabolic pathway map of grape in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and in the KaPPA-View 4 KEGG system, then projected the transcriptome and metabolome data on a metabolic pathway map. The map showed specific induction of the resveratrol synthetic pathway by UV-C light. Our results showed that multiomics is a powerful tool to elucidate the accumulation mechanisms of secondary metabolites, and updated systems, such as KEGG and KaPPA-View 4 KEGG for grape, can support such studies.

Defects in IRE1 enhance cell death and fail to degrade mRNAs encoding secretory pathway proteins in the Arabidopsis unfolded protein response.

The unfolded protein response (UPR) is a cellular response highly conserved in eukaryotes to obviate accumulation of misfolded proteins in the endoplasmic reticulum (ER). Inositol-requiring enzyme 1 (IRE1) catalyzes the cytoplasmic splicing of mRNA encoding bZIP transcription factors to activate the UPR signaling pathway. Arabidopsis IRE1 was recently shown to be involved in the cytoplasmic splicing of bZIP60 mRNA. In the present study, we demonstrated that an Arabidopsis mutant with defects in two IRE1 paralogs showed enhanced cell death upon ER stress compared with a mutant with defects in bZIP60 and wild type, suggesting an alternative function of IRE1 in the UPR. Analysis of our previous microarray data and subsequent quantitative PCR indicated degradation of mRNAs encoding secretory pathway proteins by tunicamycin, DTT, and heat in an IRE1-dependent manner. The degradation of mRNAs localized to the ER during the UPR was considered analogous to a molecular mechanism referred to as the regulated IRE1-dependent decay of mRNAs reported in metazoans. Another microarray analysis conducted in the condition repressing transcription with actinomycin D and a subsequent Gene Set Enrichment Analysis revealed the regulated IRE1-dependent decay of mRNAs-mediated degradation of a significant portion of mRNAs encoding the secretory pathway proteins. In the mutant with defects in IRE1, genes involved in the cytosolic protein response such as heat shock factor A2 were up-regulated by tunicamycin, indicating the connection between the UPR and the cytosolic protein response.

Quantitative proteomics of Arabidopsis shoot microsomal proteins reveals a cross-talk between excess zinc and iron deficiency.

Iron (Fe) deficiency significantly effects plant growth and development. Plant symptoms under excess zinc (Zn) resemble symptoms of Fe-deficient plants. To understand cross-talk between excess Zn and Fe deficiency, we investigated physiological parameters of Arabidopsis plants and applied iTRAQ-OFFGEL quantitative proteomic approach to examine protein expression changes in microsomal fraction from Arabidopsis shoots under those physiological conditions. Arabidopsis plants manifested shoot inhibition and chlorosis symptoms when grown on Fe-deficient media compared to basal MGRL solid medium. iTRAQ-OFFGEL approach identified 909 differentially expressed proteins common to all three biological replicates; the majority were transporters or proteins involved in photosynthesis, and ribosomal proteins. Interestingly, protein expression changes between excess Zn and Fe deficiency showed similar pattern. Further, the changes due to excess Zn were dramatically restored by the addition of Fe. To obtain biological insight into Zn and Fe cross-talk, we focused on transporters, where STP4 and STP13 sugar transporters were predominantly expressed and responsive to Fe-deficient conditions. Plants grown on Fe-deficient conditions showed significantly increased level of sugars. These results suggest that Fe deficiency might lead to the disruption of sugar synthesis and utilization.

Correlation analysis of proteins responsive to Zn, Mn, or Fe deficiency in Arabidopsis roots based on iTRAQ analysis.

KEY MESSAGE: For discovering the functional correlation between the identified and quantified proteins by iTRAQ analysis, here we propose a correlation analysis method with cosine correlation coefficients as a powerful tool. iTRAQ analysis is a quantitative proteomics approach that enables identification and quantification of a large number of proteins. In order to obtain proteins responsive to Zn, Mn, or Fe mineral deficiency, we conducted iTRAQ analysis using a microsomal fraction of protein extractions from Arabidopsis root tissues. We identified and quantified 730 common proteins in three biological replicates with less than 1 % false discovery rate. To determine the role of these proteins in tolerating mineral deficiencies and their relation to each other, we calculated cosine correlation coefficients and represented the outcomes on a correlation map for visual understanding of functional relations among the identified proteins. Functionally similar proteins were gathered into the same clusters. Interestingly, a cluster of proteins (FRO2, IRT1, AHA2, PDR9/ABCG37, and GLP5) highly responsive to Fe deficiency was identified, which included both known and unknown novel proteins involved in tolerating Fe deficiency. We propose that the correlation analysis with the cosine correlation coefficients is a powerful method for finding important proteins of interest to several biological processes through comprehensive data sets.

KaPPA-View4: a metabolic pathway database for representation and analysis of correlation networks of gene co-expression and metabolite co-accumulation and omics data.

Correlations of gene-to-gene co-expression and metabolite-to-metabolite co-accumulation calculated from large amounts of transcriptome and metabolome data are useful for uncovering unknown functions of genes, functional diversities of gene family members and regulatory mechanisms of metabolic pathway flows. Many databases and tools are available to interpret quantitative transcriptome and metabolome data, but there are only limited ones that connect correlation data to biological knowledge and can be utilized to find biological significance of it. We report here a new metabolic pathway database, KaPPA-View4 (http://kpv.kazusa.or.jp/kpv4/), which is able to overlay gene-to-gene and/or metabolite-to-metabolite relationships as curves on a metabolic pathway map, or on a combination of up to four maps. This representation would help to discover, for example, novel functions of a transcription factor that regulates genes on a metabolic pathway. Pathway maps of the Kyoto Encyclopedia of Genes and Genomes (KEGG) and maps generated from their gene classifications are available at KaPPA-View4 KEGG version (http://kpv.kazusa.or.jp/kpv4-kegg/). At present, gene co-expression data from the databases ATTED-II, COXPRESdb, CoP and MiBASE for human, mouse, rat, Arabidopsis, rice, tomato and other plants are available.

Functional genomics of tomato in a post-genome-sequencing phase.

Completion of tomato genome sequencing project has broad impacts on genetic and genomic studies of tomato and Solanaceae plants. The reference genome sequence derived from Solanum lycopersicum cv 'Heinz 1706' serves as the firm basis for sequencing-based approaches to tomato genomics. In this article, we first present a brief summary of the genome sequencing project and a summary of the reference genome sequence. We then focus on recent progress in transcriptome sequencing and small RNA sequencing and show how the reference genome sequence makes these analyses more comprehensive than before. We discuss the potential of in-depth analysis that is based on DNA methylome sequencing and transcription start-site detection. Finally, we describe the current status of efforts to resequence S. lycopersicum cultivars to demonstrate how resequencing can allow the use of intraspecific genomic diversity for detailed phenotyping and breeding.

Analyses of S Protein Homology Using the Genomes of SARS-CoV-2 Specimens Unveil Missing Links in the Temporal Order of Mutations in Its Variants.

(1) Background: Since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the evolutionary traits of its variants have been revealed. However, the temporal order of the majority of mutations harbored by variants after the closest ancestors (or precursors), as "missing links", remains unclear. In this study, we aimed to unveil such missing links based on analyses of S protein homology by focusing on specimens with incomplete sets of S protein mutations in a variant. (2) Methods: Prevariant and postvariant mutations were defined as those before and after the variant's development, respectively. A total of 6,758,926 and 14,519,521 genomes were obtained from the National Center for Biotechnology Information and the GISAID initiative, respectively, and S protein mutations were detected based on BLASTN analyses. (3) Results: The temporal order of prevariant mutations harbored by 12 variants was deduced. In particular, the D950N mutation in the Mu variant shows V-shaped mutation transitions, in which multiple routes of evolution were combined and resulted in the formation of a V-shaped transition, indicating recombination. (4) Conclusions: Many genome data for SARS-CoV-2 unveiled the candidate precursors of Mu variant based on a data-driven approach to its prevariant mutations in each nation.

Identification and characterization of ANAC042, a transcription factor family gene involved in the regulation of camalexin biosynthesis in Arabidopsis.

Camalexin is the major phytoalexin in Arabidopsis. An almost complete set of camalexin biosynthetic enzymes have been elucidated but only limited information is available regarding molecular mechanisms regulating camalexin biosynthesis. Here, we demonstrate that ANAC042, a member of the NAM, ATAF1/2, and CUC2 (NAC) transcription factor family genes, is involved in camalexin biosynthesis induction. T-DNA insertion mutants of ANAC042 failed to accumulate camalexin at the levels achieved in the wild type, and were highly susceptible to Alternaria brassicicola infection. The camalexin biosynthetic genes CYP71A12, CYP71A13, and CYP71B15/PAD3 were not fully induced in the mutants, indicating that the camalexin defects were at least partly a result of reduced expression levels of these P450 genes. ß-Glucuronidase (GUS)-reporter assays demonstrated tissue-specific induction of ANAC042 in response to differential pathogen infections. Bacterial flagellin (Flg22) induced ANAC042 expression in the root-elongation zone, the camalexin biosynthetic site, and the induction was abolished in the presence of either a general kinase inhibitor (K252a), a Ca(2+)-chelator (BAPTA), or methyl jasmonate. The GUS-reporter assay revealed repression of the Flg22-dependent ANAC042 expression in the ethylene-insensitive ein2-1 background but not in sid2-2 plants defective for salicylic acid biosynthesis. We discuss ANAC042 as a key transcription factor involved in previously unknown regulatory mechanisms to induce phytoalexin biosynthesis in Arabidopsis.

Construction and analysis of EST libraries of the trans-polyisoprene producing plant, Eucommia ulmoides Oliver.

Eucommia ulmoides Oliver is one of a few woody plants capable of producing abundant quantities of trans-polyisoprene rubber in their leaves, barks, and seed coats. One cDNA library each was constructed from its outer stem tissue and inner stem tissue. They comprised a total of 27,752 expressed sequence tags (ESTs) representing 10,520 unigenes made up of 4,302 contigs and 6,218 singletons. Homologues of genes coding for rubber particle membrane proteins that participate in the synthesis of high-molecular poly-isoprene in latex were isolated, as well as those encoding known major latex proteins (MLPs). MLPs extensively shared ESTs, indicating their abundant expression during trans-polyisoprene rubber biosynthesis. The six mevalonate pathway genes which are implicated in the synthesis of isopentenyl diphosphate (IPP), a starting material of poly-isoprene biosynthesis, were isolated, and their role in IPP biosynthesis was confirmed by functional complementation of suitable yeast mutants. Genes encoding five full-length trans-isoprenyl diphosphate synthases were also isolated, and two among those synthesized farnesyl diphosphate from IPP and dimethylallyl diphosphate, an assumed intermediate of rubber biosynthesis. This study should provide a valuable resource for further studies of rubber synthesis in E. ulmoides.

A database for retrieving information on SARS-CoV-2 S protein mutations based on correlation network analysis.

BACKGROUND: Over a million genomes and mutational analyses of SARS-CoV-2 are available in public databases, which reveal the phylogenetic tree of the virus. Although these data have enabled scientists to closely track the evolution and transmission dynamics of the virus at global and local scales, the Mu variant, recently identified in infections in South America, shows an unusual combination of mutations, and it is difficult to visualize these atypical characteristics in public databases based on a phylogenetic tree. RESULTS: The Vcorn SARS-CoV-2 database was constructed to provide information on COVID-19 infections and mutations in the S protein of the virus based on correlation network analysis. A correlation network was constructed using the recall index of one mutation to another mutation. The network includes several network modules in which nodes represent mutations and are tightly connected to each other. Individual network modules contain mutations of single variants, such as the alpha and delta variants. In the network constructed to emphasize mutations of the Mu variant using the database, the mutations were found to be located in multiple network modules, indicating that the mutations of the variant may have originated from multiple variants or be located at a basal position with a high frequency of mutation. CONCLUSIONS: Vcorn SARS-CoV-2 provides information on COVID-19 and S protein mutations of SARS-CoV-2 via correlation network analysis. The network based on the analysis illustrates the unusual S protein mutations of the Mu variant. The database is freely available at http://www.plant.osakafu-u.ac.jp/~kagiana/vcorn/sarscov2/ .

CoP: a database for characterizing co-expressed gene modules with biological information in plants.

UNLABELLED: Using a large dataset (10 022 assays) obtained from public plant microarray databases, we developed the CoP database for associating co-expressed gene modules with biological information such as gene ontology terms and, if available, metabolic pathway names. The Confeito algorithm developed previously in our laboratory, which is suitable to calculate the interconnectivity between genes in co-expressed gene network, was applied to extract co-expressed gene modules. The database includes the gene modules for Arabidopsis thaliana (thale cress) and seven crops, Glycine max (soybean), Hordeum vulgare (barley), Oryza sativa (rice), Populus trichocarpa (poplar), Triticum aestivum (wheat), Vitis vinifera (grape) and Zea mays (maize). AVAILABILITY: The CoP database is available at: http://webs2.kazusa.or.jp/kagiana/cop0911/.

Gcorn fungi: A Web Tool for Detecting Biases between Gene Evolution and Speciation in Fungi.

(1) Background: Fungi contain several millions of species, and the diversification of fungal genes has been achieved by speciation, gene duplication, and horizontal gene transfer. Although several databases provide information on orthologous and paralogous events, these databases show no information on biases between gene mutation and speciation. Here, we designed the Gcorn fungi database to better understand such biases. (2) Methods: Amino acid sequences of fungal genes in 249 species, which contain 2,345,743 sequences, were used for this database. Homologous genes were grouped at various thresholds of the homology index, which was based on the percentages of gene mutations. By grouping genes that showed highly similar homology indices to each other, we showed functional and evolutionary traits in the phylogenetic tree depicted for the gene of interest. (3) Results: Gcorn fungi provides well-summarized information on the evolution of a gene lineage and on the biases between gene evolution and speciation, which are quantitatively identified by the Robinson-Foulds metric. The database helps users visualize these traits using various depictions. (4) Conclusions: Gcorn fungi is an open access database that provides a variety of information with which to understand gene function and evolution.

Genetic Mapping of the HLA1 Locus Causing Hybrid Lethality in Nicotiana Interspecific Hybrids.

Hybrid lethality, a postzygotic mechanism of reproductive isolation, is a phenomenon that causes the death of F1 hybrid seedlings. Hybrid lethality is generally caused by the epistatic interaction of two or more loci. In the genus Nicotiana, N. debneyi has the dominant allele Hla1-1 at the HLA1 locus that causes hybrid lethality in F1 hybrid seedlings by interaction with N. tabacum allele(s). Here, we mapped the HLA1 locus using the F2 population segregating for the Hla1-1 allele derived from the interspecific cross between N. debneyi and N. fragrans. To map HLA1, several DNA markers including random amplified polymorphic DNA, amplified fragment length polymorphism, and simple sequence repeat markers, were used. Additionally, DNA markers were developed based on disease resistance gene homologs identified from the genome sequence of N. benthamiana. Linkage analysis revealed that HLA1 was located between two cleaved amplified polymorphic sequence markers Nb14-CAPS and NbRGH1-CAPS at a distance of 10.8 and 10.9 cM, respectively. The distance between these markers was equivalent to a 682 kb interval in the genome sequence of N. benthamiana.

Characterization of the ABA-regulated global responses to dehydration in Arabidopsis by metabolomics.

Drought is the major environmental threat to agricultural production and distribution worldwide. Adaptation by plants to dehydration stress is a complex biological process that involves global changes in gene expression and metabolite composition. Here, using one type of functional genomics analysis, metabolomics, we characterized the metabolic phenotypes of Arabidopsis wild-type and a knockout mutant of the NCED3 gene (nc3-2) under dehydration stress. NCED3 plays a role in the dehydration-inducible biosynthesis of abscisic acid (ABA), a phytohormone that is important in the dehydration-stress response in higher plants. Metabolite profiling performed using two types of mass spectrometry (MS) systems, gas chromatography/time-of-flight MS (GC/TOF-MS) and capillary electrophoresis MS (CE-MS), revealed that accumulation of amino acids depended on ABA production, but the level of the oligosaccharide raffinose was regulated by ABA independently under dehydration stress. Metabolic network analysis showed that global metabolite-metabolite correlations occurred in dehydration-increased amino acids in wild-type, and strong correlations with raffinose were reconstructed in nc3-2. An integrated metabolome and transcriptome analysis revealed ABA-dependent transcriptional regulation of the biosynthesis of the branched-chain amino acids, saccharopine, proline and polyamine. This metabolomics analysis revealed new molecular mechanisms of dynamic metabolic networks in response to dehydration stress.

Chemical genetic approach using ß-rubromycin reveals that a RIO kinase-like protein is involved in morphological development in Phytophthora infestans.

To characterize the molecular mechanisms underlying life-stage transitions in Phytophthora infestans, we initiated a chemical genetics approach by screening for a stage-specific inhibitor of morphological development from microbial culture extracts prepared mostly from actinomycetes from soil in Japan. Of the more than 700 extracts, one consistently inhibited Ph. infestans cyst germination. Purification and identification of the active compound by ESI-MS, 1H-NMR, and 13C-NMR identified ß-rubromycin as the inhibitor of cyst germination (IC50 = 19.8 µg/L); ß-rubromycin did not inhibit growth on rye media, sporangium formation, zoospore release, cyst formation, or appressorium formation in Ph. infestans. Further analyses revealed that ß-rubromycin inhibited the germination of cysts and oospores in Pythium aphanidermatum. A chemical genetic approach revealed that ß-rubromycin stimulated the expression of RIO kinase-like gene (PITG_04584) by 60-fold in Ph. infestans. Genetic analyses revealed that PITG_04584, which lacks close non-oomycete relatives, was involved in zoosporogenesis, cyst germination, and appressorium formation in Ph. infestans. These data imply that further functional analyses of PITG_04584 may contribute to new methods to suppress diseases caused by oomycetes.