Defects In Germination Research Articles

SterylAcetyl Hydrolase 1 (BbSay1) Links Lipid Homeostasis to Conidiogenesis and Virulence in the Entomopathogenic Fungus Beauveria bassiana.

Beauveria bassiana, as a well-studied entomopathogenic fungus, has a great potential for the biological control of insect pests. Lipid metabolism has been linked to the life cycle of B. bassiana; however, the underlying mechanisms remain unknown. In this study, a homolog of yeast steryl acetyl hydrolase 1 (Say1) was functionally characterized. The loss of B. bassianaSAY1 (BbSAY1) impaired the lipid homeostasis in conidia, with a significant reduction in oleic acid content. The ΔBbsay1 mutant strain displayed anelevated accumulation of lipid bodies and aweakened membrane permeability. As for phenotypic aspects, gene loss resulted in significant defects in germination, conidiation, and virulence. Our findings highlight that Say1, involved in lipid homeostasis, contributes to the cytomembrane integrity, development, and virulence in B. bassiana.

ZmCOI2a and ZmCOI2b redundantly regulate anther dehiscence and gametophytic male fertility in maize.

In higher plants, the generation and release of viable pollen from anthers is vital for double fertilization and the initiation of seed development. Thus, the characterization of genes related to pollen development and anther dehiscence in plants is of great significance. The F-box protein COI1 plays a crucial role in the jasmonate (JA) signaling pathway and interacts with many JAZ family proteins in the presence of jasmonoyl-isoleucine (JA-Ile) or coronatine (COR). The mutation of AtCOI1 in Arabidopsis leads to defective anther dehiscence and male sterility (MS), although COI has not been shown to affect fertility in Zea mays (maize). Here we identified two genes, ZmCOI2a and ZmCOI2b, that redundantly regulate gametophytic male fertility. Both ZmCOI2a and ZmCOI2b are highly homologous and constitutively expressed in all tissues tested. Subcellular localization revealed that ZmCOI2a and ZmCOI2b were located in the nucleus. The coi2a coi2b double mutant, generated by CRISPR/Cas9, had non-dehiscent anthers, delayed anther development and MS. In addition, coi2a coi2b male gametes could not be transmitted to the next generation because of severe defects in pollen germination. The JA content of coi2a coi2b anthers was unaltered compared with those of the wild type, and the exogenous application of JA could not rescue the fertility defects of coi2a coi2b. Transcriptome analysis showed that the expression of genes involving the JA signaling transduction pathway, including ZmJAZ3, ZmJAZ4, ZmJAZ5 and ZmJAZ15, was affected in coi2a coi2b. However, yeast two-hybrid assays showed that ZmJAZs interacted with ZmCOI1s, but not with ZmCOI2s. In conclusion, ZmCOI2a and ZmCOI2b redundantly regulate anther dehiscence and gametophytic male fertility in maize.

Chromatin remodeling factors OsYAF9 and OsSWC4 interact to promote internode elongation in rice.

Deposition of H2A.Z and H4 acetylation by SWI2/SNF2-Related 1 Chromatin Remodeling (SWR1) and Nucleosome Acetyltransferase of H4 (NuA4) complexes in specific regulatory regions modulates transcription and development. However, little is known about these complexes in Oryza sativa (rice) development. Here, we reported that OsYAF9 and OsSWC4, two subunits of SWR1 and NuA4 complexes, are involved in rice vegetative and reproductive development. Loss of OsYAF9 resulted in reduced height, fewer tillers, fewer pollen grains, and defects in embryogenesis and seed filling. OsYAF9 directly interacted with OsSWC4 in vitro and in vivo. Loss of OsSWC4 function exhibited defects in pollen germination and failure to generate seeds, whereas knockdown of OsSWC4 resulted in reduced height and fewer tillers. The reduced height caused by OsYAF9 mutation and OsSWC4 knockdown was due to shorter internodes and defects in cell elongation, and this phenotype was rescued with gibberellin (GA) treatment, suggesting that both OsYAF9 and OsSWC4 are involved in the GA biosynthesis pathway. OsSWC4 was directly bound to the AT-rich region of GA biosynthesis genes, which in turn accomplished H2A.Z deposition and H4 acetylation at the GA biosynthesis genes with OsYAF9. Together, our study provides insights into the mechanisms involving OsSWC4 and OsYAF9 forming a protein complex to promote rice internode elongation with H2A.Z deposition and H4 acetylation.

Aolatg1 and Aolatg13 Regulate Autophagy and Play Different Roles in Conidiation, Trap Formation, and Pathogenicity in the Nematode-Trapping Fungus Arthrobotrys oligospora.

Autophagy is a conserved cellular recycling and trafficking pathway in eukaryotes that plays an important role in cell growth, development, and pathogenicity. Atg1 and Atg13 form the Atg1–Atg13 complex, which is essential for autophagy in yeast. Here, we characterized the roles of the Aolatg1 and Aolatg13 genes encoding these autophagy-related proteins in the nematode-trapping fungus Arthrobotrys oligospora. Investigation of the autophagy process by using the AoAtg8-GFP fusion protein showed that autophagosomes accumulated inside vacuoles in the wild-type (WT) A. oligospora strain, whereas in the two mutant strains with deletions of Aolatg1 or Aolatg13, GFP signals were observed outside vacuoles. Similar results were observed by using transmission electron microscopy. Furthermore, deletion of Aolatg1 caused severe defects in mycelial growth, conidiation, conidial germination, trap formation, and nematode predation. In addition, transcripts of several sporulation-related genes were significantly downregulated in the ΔAolatg1 mutant. In contrast, except for the altered resistance to several chemical stressors, no obvious differences were observed in phenotypic traits between the WT and ΔAolatg13 mutant strains. The gene ontology analysis of the transcription profiles of the WT and ΔAolatg1 mutant strains showed that the set of differentially expressed genes was highly enriched in genes relevant to membrane and cellular components. The Kyoto Encyclopedia of Genes and Genomes analysis indicated that differentially expressed genes were highly enriched in those related to metabolic pathways, autophagy and autophagy-related processes, including ubiquitin-mediated proteolysis and SNARE interaction in vesicular transport, which were enriched during trap formation. These results indicate that Aolatg1 and Aolatg13 play crucial roles in the autophagy process in A. oligospora. Aolatg1 is also involved in the regulation of asexual growth, trap formation, and pathogenicity. Our results highlight the importance of Aolatg1 in the growth and development of A. oligospora, and provide a basis for elucidating the role of autophagy in the trap formation and pathogenicity of nematode-trapping fungi.

BOB.1/OBF.1 is required during B-cell ontogeny for B-cell differentiation and germinal center function.

BOB.1/OBF.1 is a lymphocyte-specific transcriptional co-activator of octamer-dependent transcription. It regulates the expression of genes important for lymphocyte physiology together with the Oct-1 and Oct-2 transcription factors. So far, BOB.1/OBF.1 has been studied in conventional knockout mice, whereby a function of BOB.1/OBF.1 in B but also in T cells was described. The main characteristic of BOB.1/OBF.1-deficient mice is the complete absence of germinal centers. However, it is entirely unsolved at which stage of B-cell development BOB.1/OBF.1 expression is essential for germinal center formation. Still, it is not known whether defects observed late in B-cell development of BOB.1/OBF.1-deficient mice are merely a consequence of defective early B-cell development. To answer the question, whether BOB.1/OBF.1 expression is required before or during the process of germinal center formation, we established a mouse system, which allows the conditional deletion of BOB.1/OBF.1 at different stages of B-cell development. Our data reveal a requirement for BOB.1/OBF.1 during both early antigen-independent and late antigen-dependent B-cell development, and further a requirement for efficient germinal center reaction during complete B-cell ontogeny. By specifically deleting BOB.1/OBF.1 in germinal center B cells, we provide evidence that the failure to form germinal centers is a germinal center B-cell intrinsic defect and not exclusively a consequence of defective early B-cell maturation.

Functional characterization of hydroxyproline-O-galactosyltransferases for Arabidopsis arabinogalactan-protein synthesis

BackgroundArabinogalactan-proteins (AGPs) are structurally complex hydroxyproline-rich cell wall glycoproteins ubiquitous in the plant kingdom. AGPs biosynthesis involves a series of post-translational modifications including the addition of type II arabinogalactans to non-contiguous Hyp residues. To date, eight Hyp-galactosyltransferases (Hyp-GALTs; GALT2-GALT9) belonging to CAZy GT31, are known to catalyze the addition of the first galactose residues to AGP protein backbones and enable subsequent AGP glycosylation. The extent of genetic redundancy, however, remains to be elucidated for the Hyp-GALT gene family.ResultsTo examine their gene redundancy and functions, we generated various multiple gene knock-outs, including a triple mutant (galt5 galt8 galt9), two quadruple mutants (galt2 galt5 galt7 galt8, galt2 galt5 galt7 galt9), and one quintuple mutant (galt2 galt5 galt7 galt8 galt9), and comprehensively examined their biochemical and physiological phenotypes. The key findings include: AGP precipitations with β-Yariv reagent showed that GALT2, GALT5, GALT7, GALT8 and GALT9 act redundantly with respect to AGP glycosylation in cauline and rosette leaves, while the activity of GALT7, GALT8 and GALT9 dominate in the stem, silique and flowers. Monosaccharide composition analysis showed that galactose was decreased in the silique and root AGPs of the Hyp-GALT mutants. TEM analysis of 25789 quintuple mutant stems indicated cell wall defects coincident with the observed developmental and growth impairment in these Hyp-GALT mutants. Correlated with expression patterns, galt2, galt5, galt7, galt8, and galt9 display equal additive effects on insensitivity to β-Yariv-induced growth inhibition, silique length, plant height, and pollen viability. Interestingly, galt7, galt8, and galt9 contributed more to primary root growth and root tip swelling under salt stress, whereas galt2 and galt5 played more important roles in seed morphology, germination defects and seed set. Pollen defects likely contributed to the reduced seed set in these mutants.ConclusionAdditive and pleiotropic effects of GALT2, GALT5, GALT7, GALT8 and GALT9 on vegetative and reproductive growth phenotypes were teased apart via generation of different combinations of Hyp-GALT knock-out mutants. Taken together, the generation of higher order Hyp-GALT mutants demonstrate the functional importance of AG polysaccharides decorating the AGPs with respect to various aspects of plant growth and development.

The Molecular Mechanisms of Phytophthora infestans in Response to Reactive Oxygen Species Stress.

Reactive oxygen species (ROSs) are critical for the growth, development, proliferation, and pathogenicity of microbial pathogens; however, excessive levels of ROSs are toxic. Little is known about the signaling cascades in response to ROS stress in oomycetes such as Phytophthora infestans, the causal agent of potato late blight. Here, P. infestans was used as a model system to investigate the mechanism underlying the response to ROS stress in oomycete pathogens. Results showed severe defects in sporangium germination, mycelium growth, appressorium formation, and virulence of P. infestans in response to H2O2 stress. Importantly, these phenotypes mimic those of P. infestans treated with rapamycin, the inhibitor of target of rapamycin (TOR, 1-phosphatidylinositol-3-kinase). Strong synergism occurred when P. infestans was treated with a combination of H2O2 and rapamycin, suggesting that a crosstalk exists between ROS stress and the TOR signaling pathway. Comprehensive analysis of transcriptome, proteome, and phosphorylation omics showed that H2O2 stress significantly induced the operation of the TOR-mediated autophagy pathway. Monodansylcadaverine staining showed that in the presence of H2O2 and rapamycin, the autophagosome level increased in a dosage-dependent manner. Furthermore, transgenic potatoes containing double-stranded RNA of TOR in P. infestans (PiTOR) displayed high resistance to P. infestans. Therefore, TOR is involved in the ROS response and is a potential target for control of oomycete diseases, because host-mediated silencing of PiTOR increases potato resistance to late blight.

Transcription factor UvMsn2 is important for vegetative growth, conidiogenesis, stress response, mitochondrial morphology and pathogenicity in the rice false smut fungus Ustilaginoidea virens

Transcription factors (TFs) play critical roles in the control of development and pathogenicity of phytopathogens by directly or indirectly regulating the expression of downstream genes. Here, we identified and characterized a zinc finger TF UvMsn2 in Ustilaginoidea virens, a homolog of MoMsn2 from the rice blast fungus. Heterogenous complementation assay revealed that UvMsn2 fully restored the defects of the ∆Momsn2 mutant in vegetative growth, conidiation and pathogenicity. Deletion of UvMsn2 in U. virens led to a reduction of the pathogen in vegetative growth, aerial hyphae and conidiation. Additionally, the ∆Uvmsn2 mutant displayed defects in conidial morphology and germination, as well as mitochondrial morphology. Pathogenicity and toxicity assays revealed that the ∆Uvmsn2 mutant was non-pathogenic and less inhibitory to rice seed germination. The ∆Uvmsn2 mutant showed different sensitivity to various stresses. Further microscopic examination found that UvMsn2 was localized in both cytosol and nucleus, and translocated from cytosol to nucleus under the treatment of NaCl. Our results demonstrate that UvMsn2 is a critical TF that regulates the vegetative growth, conidiogenesis, stress response, mitochondrial morphology and virulence in the rice false smut fungus.

Heat Stress Tolerance Gene FpHsp104 Affects Conidiation and Pathogenicity of Fusarium pseudograminearum.

Heat shock protein Hsp104, a homolog of the bacterial chaperone ClpB and plant Hsp100, plays an essential part in the response to heat and various chemical agents in Saccharomyces cerevisiae. However, their functions remain largely unknown in plant fungal pathogens. Here, we report the identification and functional characterization of a plausible ortholog of yeast Hsp104 in Fusarium pseudograminearum, which we termed FpHsp104. Deletion mutant of FpHsp104 displayed severe defects in the resistance of heat shock during F. pseudograminearum mycelia and conidia when exposed to extreme heat. We also found that the protein showed dynamic localization to small particles under high temperature. However, no significant differences were detected in osmotic, oxidative, or cell wall stress responses between the wild-type and Δfphsp104 strains. Quantitative real-time PCR analysis showed that FpHsp104 was upregulated in the conidia, and disruption of FpHsp104 gene resulted in defects in conidia production, morphology, and germination. The transcript levels of conidiation-related genes of FpFluG, FpVosA, FpWetA, and FpAbaA were reduced in the Δfphsp104 mutant vs. the wild-type strain, but heat-shocked mRNA splicing repair was not affected in Δfphsp104. Moreover, Δfphsp104 mutant also showed attenuated virulence, but its DON synthesis was normal. These data from the first study of Hsp104 in F. pseudograminearum strongly suggest that FpHsp104 gene is an important element in the heat tolerance, development, and pathogenicity processes of F. pseudograminearum.

The N-mannosyltransferase gene BbAlg9 contributes to cell wall integrity, fungal development and the pathogenicity of Beauveria bassiana

The mannosyltransferase Alg9 plays a vital role in N-linked protein glycosylation in Saccharomyces cerevisiae, but its function in most filamentous fungi is not clear. The present study characterized BbAlg9 (an ortholog of S. cerevisiae Alg9) in Beauveria bassiana to determine the roles of N-mannosyltransferase in biological control potential of the filamentous entomopathogenic fungus. The disruption of BbAlg9 led to slower fungal growth in media with various nutrition compositions. The conidiation of ΔBbAlg9 was less than that of the wild type from the third to the fifth day but showed no significant difference on the sixth day, suggesting that BbAlg9 affects the development of conidia rather than conidial yield of late stage. ΔBbAlg9 showed defects in conidial germination, multiple stress tolerances and the yield of blastospores, with altered size and density, and virulence in hosts infected via the immersion and injection methods. The deletion of BbAlg9 resulted in defects in cell wall integrity, including increased mannoprotein and glucan content and decreased chitin content, which were accompanied by transcriptional activation or suppression of genes related to cell wall component biosynthesis. Notably, deletion of the N-mannosyltransferase BbAlg9 altered the transcription levels of O-mannosyltransferase genes (Pmt and Ktr family). These data show that BbAlg9 is involved in the fungal development, conidial stress tolerance, cell wall integrity and virulence of B. bassiana.

Evaluation of supercritical CO2 sterilization efficacy for sanitizing personal protective equipment from the coronavirus SARS-CoV-2.

The purpose of this research is to evaluate the supercritical carbon dioxide (scCO2) sterilization-based NovaClean process for decontamination and reprocessing of personal protective equipment (PPE) such as surgical masks, cloth masks, and N95 respirators. Preliminarily, Bacillus atrophaeus were inoculated into different environments (dry, hydrated, and saliva) to imitate coughing and sneezing and serve as a “worst-case” regarding challenged PPE. The inactivation of the microbes by scCO2 sterilization with NovaKill or H2O2 sterilant was investigated as a function of exposure times ranging from 5 to 90 min with a goal of elucidating possible mechanisms. Also, human coronavirus SARS-CoV-2 and HCoV-NL63 were inoculated on the respirator material, and viral activity was determined post-treatment. Moreover, we investigated the reprocessing ability of scCO2-based decontamination using wettability testing and surface mapping. Different inactivation mechanisms have been identified in scCO2 sanitization, such as membrane damage, germination defect, and dipicolinic acid leaks. Moreover, the viral sanitization results showed a complete inactivation of both coronavirus HCoV-NL63 and SARS-CoV-2. We did not observe changes in PPE morphology, topographical structure, or material integrity, and in accordance with the WHO recommendation, maintained wettability post-processing. These experiments establish a foundational understanding of critical elements for the decontamination and reuse of PPE in any setting and provide a direction for future research in the field.

Quantitative trait locus mapping for resistance to heat‐induced seed degradation and low seed phytic acid in soybean

AbstractSoybean [Glycine max (L.) Merr.] reproductive structures are temperature‐sensitive, with a reproductive optimum of 22 to 24 °C. Currently, parts of the US soybean growing region experience consistent late‐season drought stress, resulting in the adoption of agronomic practices that favor early maturity groups. This approach is the Early Soybean Production System and has boosted yields and on‐farm returns on investment. However, seeds produced under this system develop under higher temperatures than standard practices, and frequently have decreased seed quality, loss of value, and unacceptable germination rates. Climate change may result in more widespread late‐season drought and elevated temperatures during seed filling. The ancestors of all modern US high‐yielding soybean lines lack substantial resistance to heat‐induced seed degradation, but an unadapted plant introduction (PI 587982A) can maintain ∼95% quality and germination under the same conditions. Inconsistent germination and emergence defects are associated with increased bioavailable phosphorus and reduced phytic acid. To evaluate these traits’ interaction, a backcross recombinant inbred line population was developed, and emergence and germination were evaluated in seed produced in six greenhouse and field environments. Two mutant alleles (lpa1a and lpa2a) were linked to decreased germination and emergence; this effect was pronounced under elevated temperatures. A novel major‐effect heat tolerance quantitative trait locus derived from PI 587982A was identified and found to be associated with positive effects on overall germination and emergence. These results open the potential application of marker‐assisted selection for tolerance to elevated temperatures and may accelerate the development of germplasm and cultivars with high‐quality seed.

Brassinosteroid and gibberellin coordinate rice seed germination and embryo growth by regulating glutelin mobilization

Seed germination is the beginning of a new lifecycle, and involves many complex physiological and biochemical reactions including seed reserve mobilization in the endosperm and nutrient transport and reuse in the embryo. Although glutelin is a dominant storage protein in rice, its contribution to seed germination and its regulatory mechanisms are mostly unknown. Gibberellin (GA) and brassinosteroid (BR), two major growth-promoting phytohormones, also play positive roles in controlling seed germination. However, how GA and BR interact and coordinate seed germination and facilitate glutelin mobilization remains unclear. In the present study, biochemical and physiological analyses of seed germination indicated that both GA and BR promote seed germination and post-germination growth. Exogenous application of GA restored germination defects caused by BR deficiency or insensitivity. Proteomic and qRT-PCR results showed that the expression of several glutelin proteins and their encoding genes was induced by BR and GA in the embryo. Expression assays suggested that the increased accumulation of glutelin protein in the embryo was due to the accelerated degradation of glutelin by a cysteine proteinase (REP-1) in the endosperm. The breakdown of glutelin in the endosperm showed a strict positive correspondence with the length of the shoot. The GluA2 mutation led to reduced degradation rate of glutelin and defects in seed germination, and the promotion effect of GA on seed germination was weakened in the glua2 mutant. In vitro culture assay of rice embryos showed that glutelin mobilization functioned downstream of the GA and BR pathways to promote shoot elongation. These findings suggest a mechanism that mediates crosstalk between BR and GA in co-regulating rice seed germination and embryo growth.

Analysis of Apoptosis-Related Genes Reveals that Apoptosis Functions in Conidiation and Pathogenesis of Fusarium pseudograminearum.

Apoptosis, a type of programmed cell death, plays crucial roles in various physiological processes, from development to adaptive responses. Key features of apoptosis have been verified in various fungal microbes but not yet in Fusarium species. Here, we identified 19 apoptosis-related genes in Fusarium pseudograminearum using a genome-wide survey. Expression profile analysis revealed that several apoptosis-related genes were significantly increased during conidiation and infection stages. Among these is FpBIR1, with two BIR (baculovirus inhibitor-of-apoptosis protein repeat) domains at the N-terminal end of the protein, a homolog of Saccharomyces cerevisiae BIR1, which is a unique apoptosis inhibitor. FpNUC1 is the ortholog of S. cerevisiae NUC1, which triggers AIF1- or YCA1-independent apoptosis. The functions of these two proteins were assessed by creating Δfpbir1 and Δfpnuc1 mutants via targeted gene deletion. The Δfpbir1 mutant had more cells with nuclear fragmentation and exhibited reduced conidiation, conidial formation, and infectivity. Correspondingly, the Δfpnuc1 mutant contained multiple nuclei, produced thicker and more branched hyphae, was reduced in conidiation, and exhibited faster conidial formation and higher infection rates. Taken together, our results indicate that the apoptosis-related genes FpBIR1 and FpNUC1 function in conidiation, conidial germination, and infection by F. pseudograminearumIMPORTANCE The plant-pathogenic fungus F. pseudograminearum is the causal agent of Fusarium crown rot (FCR) in wheat and barley, resulting in substantial yield losses worldwide. Particularly, in the Huanghuai wheat-growing region of China, F. pseudograminearum was reported as the dominant Fusarium species in FCR infections. Apoptosis is an evolutionarily conserved mechanism in eukaryotes, playing crucial roles in development and cell responses to biotic and abiotic stresses. However, few reports on apoptosis in plant fungal pathogens have been published. In this study, we identified 19 conserved apoptosis-related genes in F. pseudograminearum, several of which were significantly increased during conidiation and infection stages. Potential apoptosis functions were assessed by deletion of the putative apoptosis inhibitor gene FpBIR1 and apoptosis trigger gene FpNUC1 in F. pseudograminearum The FpBIR1 deletion mutant exhibited defects in conidial germination and pathogenicity, whereas the FpNUC1 deletion mutant experienced faster conidial formation and higher infection rates. Apoptosis appears to negatively regulate the conidial germination and pathogenicity of F. pseudograminearum To our knowledge, this study is the first report of apoptosis contributing to infection-related morphogenesis and pathogenesis in F. pseudograminearum.

OsCER1 regulates humidity-sensitive genic male sterility through very-long-chain (VLC) alkane metabolism of tryphine in rice

Humidity-sensitive genic male sterility (HGMS) is a novel type of environment-sensitive male sterility (EGMS) which plants are male sterile at low humidity and male fertile at high humidity. Previous studies have revealed that OsCER1 contributes to very-long-chain (VLC) alkanes biosynthesis in rice (Oryza sativa L.). Here, applying the CRISPR/Cas9 technique, we obtained two independent OsCER1 knockout lines (OsCER1Cas). Both OsCER1Cas lines exhibited HGMS. Mutant pollen showed defects in adhesion and germination on stigmas at low humidity, whereas high humidity enhanced the pollen germination rate. Transmission electron microscopy (TEM) observations of mutant pollen revealed abnormal tryphine structure, potentially representing the basis of HGMS. Furthermore, co-pollination with mixed OsCER1Cas mutant and maize (Zea mays L.) pollen could rescue the fertility of the mutant, thereby establishing the key role of tryphine in germination on stigmas. OsCER1 knockout might affect VLC alkane metabolism and therefore alter the lipid composition of tryphine. It could lead to the defects in pollen grain adhesion, hydration and germination, resulting in HGMS. This work identified the mechanism of HGMS induced by VLC alkanes in rice and the generality of tryphine in different species of Gramineae.

The Dual Function of OsSWEET3a as a Gibberellin and Glucose Transporter Is Important for Young Shoot Development in Rice.

Translocation and long-distance transport of phytohormones are considered important processes for phytohormone responses, as well as their synthesis and signaling. Here, we report on the dual function of OsSWEET3a, a bidirectional sugar transporter from clade I of the rice SWEET family of proteins, as both a gibberellin (GA) and a glucose transporter. OsSWEET3a efficiently transports GAs in the C13-hydroxylation pathway of GA biosynthesis. Both knockout and overexpression lines of OsSWEET3a showed defects in germination and early shoot development, which were partially restored by GA, especially GA20. Quantitative reverse transcription PCR, GUS staining and in situ hybridization revealed that OsSWEET3a was expressed in vascular bundles in basal parts of the seedlings. OsSWEET3a expression was co-localized with OsGA20ox1 expression in the vascular bundles but not with OsGA3ox2, whose expression was restricted to leaf primordia and young leaves. These results suggest that OsSWEET3a is expressed in the vascular tissue of basal parts of seedlings and is involved in the transport of both GA20 and glucose to young leaves, where GA20 is possibly converted to the bioactive GA1 form by OsGA3ox2, during early plant development. We also indicated that such GA transport activities of SWEET proteins have sporadically appeared in the evolution of plants: GA transporters in Arabidopsis have evolved from sucrose transporters, while those in rice and sorghum have evolved from glucose transporters.

Rho GTPase ROP1 Interactome Analysis Reveals Novel ROP1-Associated Pathways for Pollen Tube Polar Growth in Arabidopsis.

ROP (Rho-like GTPases from plants) GTPases are polarly localized key regulators of polar growth in pollen tubes and other cells in plants. However, how ROP GTPases are regulated and how they control polar growth remains to be fully understood. To gain new insights into ROP-dependent mechanisms underlying polar cell growth, we characterized the interactome of ROP1 GTPase that controls Arabidopsis pollen tube (PT) tip growth, an extreme form of polar cell growth. We established an efficient method for culturing Arabidopsis pollen tubes in liquid medium, which was used for immunoprecipitation/mass spectrometry-based identification of ROP1-associated proteins. A total of 654 candidates were isolated from the ROP1 interactome in Arabidopsis pollen tubes, and GO (Gene Ontology) classification and pathway analysis revealed multiple uncharacterized ROP1-dependent processes including translation, cell wall modification, post transcriptional modification, and ion homeostasis, in addition to known ROP1-dependent pathways. The ROP1-interactome data was further supported by the co-expression of the candidate interactors in highly mature pollen with PT germination and growth defects being discovered in 25% (8/32) of the candidate mutant genes. Taken together, our work uncovers valuable information for the identification and functional elucidation of ROP-associated proteins in the regulation of polar growth, and provides a reliable reference to identify critical regulators of polar cell growth in the future.

Cooperation and Cheating among Germinating Spores

Many microbes produce stress-resistant spores to survive unfavorable conditions [1-4] and enhance dispersal [1, 5]. Cooperative behavior is integral to the process of spore formation in some species [3, 6], but the degree to which germination of spore populations involves social interactions remains little explored. Myxococcus xanthus is a predatory soil bacterium that upon starvation forms spore-filled multicellular fruiting bodies that often harbor substantial diversity of endemic origin [7, 8]. Here we demonstrate that germination of M.xanthus spores formed during fruiting-body development is a social process involving at least two functionally distinct social molecules. Using pairs of natural isolates each derived from a single fruiting body that emerged on soil, we first show that spore germination exhibits positive density dependence due to a secreted "public-good" germination factor. Further, we find that a germination defect of one strain under saline stress in pure culture is complemented by addition of another strain that germinates well in saline environments and mediates cheating by the defective strain. Glycine betaine, an osmo-protectant utilized in all domains of life, is found to mediate saline-specific density dependence and cheating. Density dependence in non-saline conditions is mediated by a distinct factor, revealing socially complex spore germination involving multiple social molecules.

A Histone Deacetylase, MoHDA1 Regulates Asexual Development and Virulence in the Rice Blast Fungus.

Interplay between histone acetylation and deacetylation is one of the key components in epigenetic regulation of transcription. Here we report the requirement of Mo-HDA1-mediated histone deacetylation during asexual development and pathogenesis for the rice blast fungus, Magnaporthe oryzae. Structural similarity and phylogenetic analysis suggested that MoHDA1 is an ortholog of Saccharomyces cerevisiae Hda1, which is a representative member of class II histone deacetylases. Targeted deletion of MoHDA1 caused a little decrease in radial growth and large reduction in asexual sporulation. Comparison of acetylation levels for H3K9 and H3K14 showed that lack of MoHDA1 gene led to significant increase in H3K9 and H3K14 acetylation level, compared to the wild-type and complementation strain, confirming that it is a bona fide histone deacetylase. Expression analysis on some of the key genes involved in asexual reproduction under sporulation-promoting condition showed almost no differences among strains, except for MoCON6 gene, which was up-regulated more than 6-fold in the mutant than wild-type. Although the deletion mutant displayed little defects in germination and subsequent appressorium formation, the mutant was compromised in its ability to cause disease. Woundinoculation showed that the mutant is impaired in invasive growth as well. We found that the mutant was defective in appressorium-mediated penetration of host, but did not lose the ability to grow on the media containing H2O2. Taken together, our data suggest that MoHDA1-dependent histone deacetylation is important for efficient asexual development and infection of host plants in M. oryzae.

Crosstalk Between Pheromone Signaling and NADPH Oxidase Complexes Coordinates Fungal Developmental Processes.

Sexual and asexual development in filamentous ascomycetes is controlled by components of conserved signaling pathways. Here, we investigated the development of mutant strains lacking genes for kinases MAK2, MEK2, and MIK2, as well as the scaffold protein HAM5 of the pheromone response (PR) pathway. All had a defect in fruiting body development and hyphal fusion. Another phenotype was a defect in melanin-dependent ascospore germination. However, this deficiency was observed only in kinase deletion mutants, but not in strains lacking HAM5. Notably, the same developmental phenotypes were previously described for nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 (NOX1) mutants, but the germination defect was only seen in NOX2 mutants. These data suggest a molecular link between the pheromone signaling pathway and both NOX complexes. Using data from yeast two-hybrid (Y2H) analysis, we found that the scaffolding protein HAM5 interacts with NOR1, the regulator of NOX1 and NOX2 complexes. This interaction was further confirmed using differently fluorescent-labeled proteins to demonstrate that NOR1 and HAM5 co-localize at cytoplasmic spots and tips of mature hyphae. This observation was supported by phenotypic characterization of single and double mutants. The oxidative stress response and the initiation of fruiting bodies were similar in Δham5Δnor1 and Δham5, but distinctly reduced in Δnor1, indicating that the double deletion leads to a partial suppression of the Δnor1 phenotype. We conclude that the PR and NOX1 complexes are connected by direct interaction between HAM5 and NOR1. In contrast, PR kinases are linked to the NOX2 complex without participation of HAM5.