Plant Cytoplasm Research Articles

Lipase domain effector AGLIP1 in Rhizoctonia solani triggers necrotic killing in plants.

We highlight the emerging role of the R. solani novel lipase domain effector AGLIP1 in suppressing pattern-triggered immunity and inducing plant cell death. The dynamic interplay between plants and Rhizoctonia solani constitutes a multifaceted struggle for survival and dominance. Within this complex dynamic, R. solani has evolved virulence mechanisms by secreting effectors that disrupt plants' first line of defense. A newly discovered effector, AGLIP1 in R. solani, plays a pivotal role in inducing plant cell death and subverting immune responses. AGLIP1, a protein containing a signal peptide and a lipase domain, involves complex formation in the intercellular space, followed by translocation to the plant cytoplasm, where it induces cell death (CD) and suppresses defense gene regulation. This study provides valuable insights into the intricate molecular interactions between plants and necrotrophic fungi, underscoring the imperative for further exploration in this field.

The Pseudomonas syringae pv. tomato DC3000 effector HopD1 interferes with cellular dynamics associated with the function of the plant immune protein AtNHR2B

The plant pathogenic bacterium Pseudomonas syringae pv tomato DC3000 (Pst DC3000) causes disease in tomato, in the model plant Arabidopsis thaliana, and conditionally in Nicotiana benthamiana. The pathogenicity of Pst DC3000 is mostly due to bacterial virulence proteins, known as effectors, that are translocated into the plant cytoplasm through the type III secretion system (T3SS). Bacterial type III secreted effectors (T3SEs) target plants physiological processes and suppress defense responses to enable and support bacterial proliferation. The Pst DC3000 T3SE HopD1 interferes with plant defense responses by targeting the transcription factor NTL9. This work shows that HopD1 also targets the immune protein AtNHR2B (Arabidopsis thaliana nonhost resistance 2B), a protein that localizes to dynamic vesicles of the plant endomembrane system. Live-cell imaging of Nicotiana benthamiana plants transiently co-expressing HopD1 fused to the epitope haemagglutinin (HopD1-HA) with AtNHR2B fused to the red fluorescent protein (AtNHR2B-RFP), revealed that HopD1-HA interferes with the abundance and cellular dynamics of AtNHR2B-RFP-containing vesicles. The results from this study shed light into an additional function of HopD1 while contributing to understanding how T3SEs also target vesicle trafficking-mediated processes in plants.

Clathrin-mediated endocytosis facilitates the internalization of Magnaporthe oryzae effectors into rice cells.

Fungi and oomycetes deliver effectors into living plant cells to suppress defenses and control plant processes needed for infection. Little is known about the mechanism by which these pathogens translocate effector proteins across the plasma membrane into the plant cytoplasm. The blast fungus Magnaporthe oryzae secretes cytoplasmic effectors into a specialized biotrophic interfacial complex (BIC) before translocation. Here, we show that cytoplasmic effectors within BICs are packaged into punctate membranous effector compartments that are occasionally observed in the host cytoplasm. Live cell imaging with fluorescently labeled proteins in rice (Oryza sativa) showed that these effector puncta colocalize with the plant plasma membrane and with CLATHRIN LIGHT CHAIN 1, a component of clathrin-mediated endocytosis (CME). Inhibiting CME using virus-induced gene silencing and chemical treatments resulted in cytoplasmic effectors in swollen BICs lacking effector puncta. By contrast, fluorescent marker colocalization, gene silencing, and chemical inhibitor studies failed to support a major role for clathrin-independent endocytosis in effector translocation. Effector localization patterns indicated that cytoplasmic effector translocation occurs underneath appressoria before invasive hyphal growth. Taken together, this study provides evidence that cytoplasmic effector translocation is mediated by CME in BICs and suggests a role for M. oryzae effectors in coopting plant endocytosis.

Comparative transcriptome analysis provides insight into the important pathways and key genes related to the pollen abortion in the thermo-sensitive genic male sterile line 373S in Brassica napus L.

The thermo-sensitive genic male sterility (TGMS) system plays a key role in the production of two-line hybrids in rapeseed (Brassica napus). To uncover key cellular events and genetic regulation associated with TGMS, a combined study using cytological methods and RNA-sequencing analysis was conducted for the rapeseed TGMS line 373S. Cytological studies showed that microspore cytoplasm of 373S plants was condensed, the microspore nucleus was degraded at an early stage, the exine was irregular, and the tapetum developed abnormally, eventually leading to male sterility. RNA-sequencing analysis identified 430 differentially expressed genes (298 upregulated and 132 downregulated) between the fertile and sterile samples. Gene ontology analysis demonstrated that the most highly represented biological processes included sporopollenin biosynthetic process, pollen exine formation, and extracellular matrix assembly. Kyoto encyclopedia of genes and genomes analysis indicated that the enriched pathways included amino acid metabolism, carbohydrate metabolism, and lipid metabolism. Moreover, 26 transcript factors were identified, which may be associated with abnormal tapetum degeneration and exine formation. Subsequently, 19 key genes were selected, which are considered to regulate pollen development and even participate in pollen exine formation. Our results will provide important insight into the molecular mechanisms underlying TGMS in rapeseed.

Role of Acrostyle Cuticular Proteins in the Retention of an Aphid Salivary Effector

To avoid the activation of plant defenses and ensure sustained feeding, aphids are assumed to use their mouthparts to deliver effectors into plant cells. A recent study has shown that effectors detected near feeding sites are differentially distributed in plant tissues. However, the precise process of effector delivery into specific plant compartments is unknown. The acrostyle, a cuticular organ located at the tip of maxillary stylets that transiently binds plant viruses via its stylin proteins, may participate in this specific delivery process. Here, we demonstrate that Mp10, a saliva effector released into the plant cytoplasm during aphid probing, binds to the acrostyles of Acyrthosiphon pisum and Myzus persicae. The effector probably interacts with Stylin-03 as a lowered Mp10-binding to the acrostyle was observed upon RNAi-mediated reduction in Stylin-03 production. In addition, Stylin-03 and Stylin-01 RNAi aphids exhibited changes in their feeding behavior as evidenced by electrical penetration graph experiments showing longer aphid probing behaviors associated with watery saliva release into the cytoplasm of plant cells. Taken together, these data demonstrate that the acrostyle also has effector binding capacity and supports its role in the delivery of aphid effectors into plant cells.

Cloning and functional characterization of lignan glycosyltransferase gene IiUGT349 in Isatis indigotica

Based on the transcriptome data of Isatis indigotica, a total of 110 putative glycosytransferases were identified. Through prokaryotic expression and enzymic activity assay in vitro, a novel lignan glycosyltransferase gene was screened out and named IiUGT349, which catalyzed lariciresinol into lariciresinol-4-O-β-D-glucoside and lariciresinol-4'-O-β-D-glucoside. Bioinformatics analysis suggested that IiUGT349 contained an open reading frame(ORF) of 1 401 bp encoding a protein of 467 amino acids. A protein analysis indicated that IiUGT349 have a predecited molecular weight of 52.77 kDa and pI of 5.96. Phylogenetic analysis showed that IiUGT349 belonging to UGT90 family shared low amino acid sequence identity with the reported lignan glycosyltransferases, which may represent a novel type of lignan glycosyltransferases. Quantitative real-time PCR(qRT-PCR) analysis showed that IiUGT349 was expressed in roots, stems, young leaves and leaves, with the highest expression level in stems. Further biochemical analysis showed that the optimal reaction time of IiUGT349 recombinant protein was 12 h and the optimal temperature was 45 ℃. Subcellular localization demonstrated that IiUGT349 was located in the cytoplasm and nucleus of plants. In this study, a new glucosyltransferase gene IiUGT349 from I. indigotica belonging to the UGT90 family was cloned, which laid a foundation to further investigate its' function and elucidate the lignan glycosides biosynthesis pathway and plays an important role for great significance for the synthetic biology of active lignan glycosides.

Priming of Solanum Tuberosum in vitro plants with PVY-specific interfering RNAs activates anti-viral resistance

Helper component-proteinase (HC-Pro) is a multifunctional suppressor protein synthesized by potato virus Y (PVY), which is able to neutralize the protective mechanisms of RNA interference (RNA-i) in potato plants, thereby causing systemic infection of the host plant and significant damage to the tuber material. This article demonstrates how one of the main functions of HC-Pro, the capture and retention of short interfering RNAs, 21-23 nt in size (siRNA) on the surface of protein subunits, can be used to create virus-resistant potato plants in vitro. The proposed method is based on the selective dissociation of the HC-Pro/siRNA complex from PVY-infected plants and siRNA-priming of healthy plants. Purified preparations of PVY-specific siRNAs were obtained using column gel filtration followed by immunoprecipitation and phenol-chloroform extraction. Injections of siRNA into the leaf plate of virus-free potato microplants, cultivars bred at Amanzholov University, led to a decrease in the accumulation of viral particles in the cytoplasm of plants inoculated with the wild PVY strain and the formation of resistance to PVY for the entire growing season.

Comprehensive prediction of plant cytoplasmic and apoplastic effectors underlying Erwinia psidii pathogenicity

AbstractErwinia psidii (Eps) is the causal agent of emerging diseases of eucalypt and guava; however, the mechanisms underlying its pathogenicity are not fully understood. Here, we predicted factors involved in the ability of Eps to cause disease on its host plants. For that, the genomes of four Eps strains exhibiting different virulence on eucalypt were sequenced, and hrp/hrc genes coding for the type III secretion system (T3SS), effectors injected into the plant cell cytoplasm through the T3SS (T3SEs) and their plant subcellular localizations, as well as proteins deployed to the host apoplast, were predicted. It was found that Eps possesses a complete hrp/hrc gene cluster based on comparison with Erwinia amylovora. A total of 18 T3SEs were predicted, 11 of which were shared among all strains, none were exclusive to any strain and seven were absent in at least one strain. No sequence variation among strains was found for five T3SE candidates whereas extensive variation was found for six, suggesting the latter may be determinants of virulence differences. The T3SE candidates are predicted to target the plant cell nucleus, cytoplasm, mitochondrion, chloroplast and peroxisome. The predicted apoplastic effector repertoire common to all four strains was over‐represented in proteins of unknown functions or predicted to possess enzymatic activities, among which the most abundant were oxidoreductases and peptidases. Proteins with lytic transglycosylase activity were predicted in strain‐specific apoplastic effector repertoires. These results provide an important framework for future research aimed at uncovering the factors underlying Eps pathogenicity.

A secreted fungal subtilase interferes with rice immunity via degradation of SUPPRESSOR OF G2 ALLELE OF skp1.

Serine protease subtilase, found widely in both eukaryotes and prokaryotes, participates in various biological processes. However, how fungal subtilase regulates plant immunity is a major concern. Here, we identified a secreted fungal subtilase, UvPr1a, from the rice false smut (RFS) fungus Ustilaginoidea virens. We characterized UvPr1a as a virulence effector localized to the plant cytoplasm that inhibits plant cell death induced by Bax. Heterologous expression of UvPr1a in rice (Oryza sativa) enhanced plant susceptibility to rice pathogens. UvPr1a interacted with the important rice protein SUPPRESSOR OF G2 ALLELE OF skp1 (OsSGT1), a positive regulator of innate immunity against multiple rice pathogens, degrading OsSGT1 in a protease activity-dependent manner. Furthermore, host-induced gene silencing of UvPr1a compromised disease resistance of rice plants. Our work reveals a previously uncharacterized fungal virulence strategy in which a fungal pathogen secretes a subtilase to interfere with rice immunity through degradation of OsSGT1, thereby promoting infection. These genetic resources provide tools for introducing RFS resistance and further our understanding of plant-pathogen interactions.

Comprehensive Effects of Flowering Locus T-Mediated Stem Growth in Tobacco.

In flowering plants, Flowering locus T (FT) encodes a major florigen. It is a key flowering hormone in controlling flowering time and has a wide range of effects on plant development. Although the mechanism by which FT promotes flowering is currently clearly understood, comprehensive effects of the FT gene on plant growth have not been evaluated. Therefore, the effects of FT on vegetative growth need to be explored for a complete understanding of the molecular functions of the FT gene. In this study, the Jatropha curcas L. FT gene was overexpressed in tobacco (JcFTOE) in order to discover multiple aspects and related mechanisms of how the FT gene affects plant development. In JcFTOE plants, root, stem, and leaf development was strongly affected. Stem tissues were selected for further transcriptome analysis. In JcFTOE plants, stem growth was affected because of changes in the nucleus, cytoplasm, and cell wall. In the nucleus of JcFTOE plants, the primary effect was to weaken all aspects of DNA replication, which ultimately affected the cell cycle and cell division. The number of stem cells decreased significantly in JcFTOE plants, which decreased the thickness and height of tobacco stems. In the cell wall of JcFTOE plants, hemicellulose and cellulose contents increased, with the increase in hemicellulose associated with up-regulation of xylan synthase-related genes expression. In the cytoplasm of JcFTOE plants, the primary effects were on biogenesis of ribonucleoprotein complexes, photosynthesis, carbohydrate biosynthesis, and the cytoskeleton. In addition, in the cytoplasm of JcFTOE plants, there were changes in certain factors of the core oscillator, expression of many light-harvesting chlorophyll a/b binding proteins was down-regulated, and expression of fructose 1,6-bisphosphatase genes was up-regulated to increase starch content in tobacco stems. Changes in the xylem and phloem of JcFTOE plants were also identified, and in particular, xylem development was affected by significant increases in expression of irregular xylem genes.

Ran-GTP/-GDP-dependent nuclear accumulation of NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 and TGACG-BINDING FACTOR2 controls salicylic acid-induced leaf senescence.

Leaf senescence is the final stage of leaf development and can be triggered by various external factors, such as hormones and light deprivation. In this study, we demonstrate that the overexpression of the GTP-bound form of Arabidopsis (Arabidopsis thaliana) Ran1 (a Ras-related nuclear small G-protein, AtRan1) efficiently promotes age-dependent and dark-triggered leaf senescence, while Ran-GDP has the opposite effect. Transcriptome analysis comparing AtRan1-GDP- and AtRan1-GTP-overexpressing transgenic plants (Ran1T27Nox and Ran1G22Vox, respectively) revealed that differentially expressed genes (DEGs) related to the senescence-promoting hormones salicylic acid (SA), jasmonic acid, abscisic acid, and ethylene (ET) were significantly upregulated in dark-triggered senescing leaves of Ran1G22Vox, indicating that these hormones are actively involved in Ran-GTP/-GDP-dependent, dark-triggered leaf senescence. Bioinformatic analysis of the promoter regions of DEGs identified diverse consensus motifs, including the bZIP motif, a common binding site for TGACG-BINDING FACTOR (TGA) transcription factors. Interestingly, TGA2 and its interactor, NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1), which are two positive transcriptional regulators of SA signaling, differed in their extent of accumulation in the nucleus versus cytoplasm of Ran1T27Nox and Ran1G22Vox plants. Moreover, SA-induced, Ran-GTP-/-GDP-dependent functions of NPR1 included genome-wide global transcriptional reprogramming of genes involved in cell death, aging, and chloroplast organization. Furthermore, the expression of AtRan1-GTP in SA signaling-defective npr1 and SA biosynthesis-deficient SA-induction deficient2 genetic backgrounds abolished the effects of AtRan1-GTP, thus retarding age-promoted leaf senescence. However, ET-induced leaf senescence was not mediated by Ran machinery-dependent nuclear shuttling of ETHYLENE-INSENSITIVE3 and ETHYLENE-INSENSITIVE3-LIKE1 proteins. We conclude that Ran-GTP/-GDP-dependent nuclear accumulation of NPR1 and TGA2 represents another regulatory node for SA-induced leaf senescence.

The Pseudomonas syringae type III effector HopG1 triggers necrotic cell death that is attenuated by AtNHR2B

The plant pathogenic bacterium Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) has become a paradigm to investigate plant-bacteria interactions due to its ability to cause disease in the model plant Arabidopsis thaliana. Pst DC3000 uses the type III secretion system to deliver type III secreted effectors (T3SEs) directly into the plant cytoplasm. Pst DC3000 T3SEs contribute to pathogenicity by suppressing plant defense responses and targeting plant’s physiological processes. Although the complete repertoire of effectors encoded in the Pst DC3000 genome have been identified, the specific function for most of them remains to be elucidated. Among those effectors, the mitochondrial-localized T3E HopG1, suppresses plant defense responses and promotes the development of disease symptoms. Here, we show that HopG1 triggers necrotic cell death that enables the growth of adapted and non-adapted pathogens. We further showed that HopG1 interacts with the plant immunity-related protein AtNHR2B and that AtNHR2B attenuates HopG1- virulence functions. These results highlight the importance of HopG1 as a multi-faceted protein and uncover its interplay with AtNHR2B.

EffectorP 3.0: Prediction of Apoplastic and CytoplasmicEffectors in Fungi and Oomycetes.

Many fungi and oomycete species are devasting plant pathogens. These eukaryotic filamentous pathogens secrete effector proteins to facilitate plant infection. Fungi and oomycete pathogens have diverse infection strategies and their effectors generally do not share sequence homology. However, they occupy similar host environments, either the plant apoplast or plant cytoplasm, and, therefore, may share some unifying properties based on the requirements of these host compartments. Here, we exploit these biological signals and present the first classifier (EffectorP 3.0) that uses two machine-learning models: one trained on apoplastic effectors and one trained on cytoplasmic effectors. EffectorP 3.0 accurately predicts known apoplastic and cytoplasmic effectors in fungal and oomycete secretomes with low estimated false-positive rates of 3 and 8%, respectively. Cytoplasmic effectors have a higher proportion of positively charged amino acids, whereas apoplastic effectors are enriched for cysteine residues. The combination of fungal and oomycete effectors in training leads to a higher number of predicted cytoplasmic effectors in biotrophic fungi. EffectorP 3.0 expands predicted effector repertoires beyond small, cysteine-rich secreted proteins in fungi and RxLR-motif containing secreted proteins in oomycetes. We show that signal peptide prediction is essential for accurate effector prediction, because EffectorP 3.0 recognizes a cytoplasmic signal also in intracellular, nonsecreted proteins.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

A tau class GST, OsGSTU5, interacts with VirE2 and modulates the Agrobacterium-mediated transformation in rice.

OsGSTU5 interacts and glutathionylates the VirE2 protein of Agrobacterium and its (OsGSTU5) overexpression and downregulation showed a low and high AMT efficiency in rice, respectively. During Agrobacterium-mediated transformation (AMT), T-DNA along with several virulence proteins such as VirD2, VirE2, VirE3, VirD5, and VirF enter the plant cytoplasm. VirE2 serves as a single-stranded DNA binding (SSB) protein that assists the cytoplasmic trafficking of T-DNA inside the host cell. Though the regulatory roles of VirE2 have been established, the cellular reaction of their host, especially in monocots, has not been characterized in detail. This study identified a cellular interactor of VirE2 from the cDNA library of rice. The identified plant protein encoded by the gene cloned from rice was designated OsGSTU5, it interacted specifically with VirE2 in the host cytoplasm. OsGSTU5 was upregulated during Agrobacterium infection and involved in the post-translational glutathionylation of VirE2 (gVirE2). Interestingly, the in silico analysis showed that the 'gVirE2 + ssDNA' complex was structurally less stable than the 'VirE2 + ssDNA' complex. The gel shift assay also confirmed the attenuated SSB property of gVirE2 over VirE2. Moreover, knock-down and overexpressionof OsGSTU5 in rice showed increased and decreased T-DNA expression, respectively after Agrobacterium infection. The present finding establishes the role of OsGSTU5 as an important target for modulation of AMT efficiency in rice.

Identification and Characterization of Wall-Associated Kinase (WAK) and WAK-like (WAKL) Gene Family in Juglans regia and Its Wild Related Species Juglans mandshurica.

Wall-associated kinase (WAK) and WAK-like kinase (WAKL) are receptor-like kinases (RLKs), which play important roles in signal transduction between the cell wall and the cytoplasm in plants. WAK/WAKLs have been studied in many plants, but were rarely studied in the important economic walnut tree. In this study, 27 and 14 WAK/WAKL genes were identified in Juglans regia and its wild related species Juglans mandshurica, respectively. We found tandem duplication might play a critical role in the expansion of WAK/WAKL gene family in J. regia, and most of the WAK/WAKL homologous pairs underwent purified selection during evolution. All WAK/WAKL proteins have the extracellular WAK domain and the cytoplasmic protein kinase domain, and the latter was more conserved than the former. Cis-acting elements analysis showed that WAK/WAKL might be involved in plant growth and development, plant response to abiotic stress and hormones. Gene expression pattern analysis further indicated that most WAK/WAKL genes in J. regia might play a role in the development of leaves and be involved in plant response to biotic stress. Our study provides a new perspective for the evolutionary analysis of gene families in tree species and also provides potential candidate genes for studying WAK/WAKL gene function in walnuts.

Plant-Mediated RNA Interference Expressing dsRNA in Cytoplasm for RNAi-Based Pest Control.

Currently, RNAi-based techniques have exhibited considerable potential for pest control, and considerable progress has been made in identifying a large number of potential target genes in the laboratory. However, applying potential target genes effectively in the field to control pests requires further research. At present, transgenic crops expressing dsRNA are widely recognized as the most practical and economical means of applying RNAi-based techniques using laboratory-identified target genes to control pests in the field. Among the three plant-mediated RNAi techniques, plant-mediated RNA interference expressing dsRNA in cytoplasm is most often considered to be an economical and practical RNAi method for pest control, both in the laboratory and inthe field. In this chapter, I describe the procedure for expressing dsRNA in plant cytoplasm for RNAi-based pest control.

Biosynthesis Pathways, Transport Mechanisms and Biotechnological Applications of Fungal Siderophores.

Iron (Fe) is the fourth most abundant element on earth and represents an essential nutrient for life. As a fundamental mineral element for cell growth and development, iron is available for uptake as ferric ions, which are usually oxidized into complex oxyhydroxide polymers, insoluble under aerobic conditions. In these conditions, the bioavailability of iron is dramatically reduced. As a result, microorganisms face problems of iron acquisition, especially under low concentrations of this element. However, some microbes have evolved mechanisms for obtaining ferric irons from the extracellular medium or environment by forming small molecules often regarded as siderophores. Siderophores are high affinity iron-binding molecules produced by a repertoire of proteins found in the cytoplasm of cyanobacteria, bacteria, fungi, and plants. Common groups of siderophores include hydroxamates, catecholates, carboxylates, and hydroximates. The hydroxamate siderophores are commonly synthesized by fungi. L-ornithine is a biosynthetic precursor of siderophores, which is synthesized from multimodular large enzyme complexes through non-ribosomal peptide synthetases (NRPSs), while siderophore-Fe chelators cell wall mannoproteins (FIT1, FIT2, and FIT3) help the retention of siderophores. S. cerevisiae, for example, can express these proteins in two genetically separate systems (reductive and nonreductive) in the plasma membrane. These proteins can convert Fe (III) into Fe (II) by a ferrous-specific metalloreductase enzyme complex and flavin reductases (FREs). However, regulation of the siderophore through Fur Box protein on the DNA promoter region and its activation or repression depend primarily on the Fe availability in the external medium. Siderophores are essential due to their wide range of applications in biotechnology, medicine, bioremediation of heavy metal polluted environments, biocontrol of plant pathogens, and plant growth enhancement.

Molecular Basis for the Substrate Promiscuity of Isopentenyl Phosphate Kinase from Candidatus methanomethylophilus alvus.

Isopentenyl phosphate kinases (IPKs) catalyze the ATP-dependent phosphorylation of isopentenyl monophosphate (IP) to isopentenyl diphosphate (IPP) in the alternate mevalonate pathways of the archaea and plant cytoplasm. In recent years, IPKs have also been employed in artificial biosynthetic pathways called "(iso) prenol pathways" that utilize promiscuous kinases to sequentially phosphorylate (iso) prenol and generate the isoprenoid precursors IPP and dimethylallyl diphosphate (DMAPP). Furthermore, IPKs have garnered attention for their impressive substrate promiscuity toward non-natural alkyl-monophosphates (alkyl-Ps), which has prompted their utilization as biocatalysts for the generation of novel isoprenoids. However, none of the IPK crystal structures currently available contain non-natural substrates, leaving the roles of active-site residues in substrate promiscuity ambiguous. To address this, we present herein the high-resolution crystal structures of an IPK from Candidatus methanomethylophilus alvus (CMA) in the apo form and bound to natural and non-natural substrates. Additionally, we describe active-site engineering studies leading to enzyme variants with broadened substrate scope, as well as structure determination of two such variants (Ile74Ala and Ile146Ala) bound to non-natural alkyl-Ps. Collectively, our crystallographic studies compare six structures of CMA variants in different ligand-bound forms and highlight contrasting structural dynamics of the two substrate-binding sites. Furthermore, the structural and mutational studies confirm a novel role of the highly conserved DVTGG motif in catalysis, both in CMA and in IPKs at large. As such, the current study provides a molecular basis for the substrate-binding modes and catalytic performance of CMA toward the goal of developing IPKs into useful biocatalysts.

A Novel Core Effector Vp1 Promotes Fungal Colonization and Virulence of Ustilago maydis

The biotrophic fungus Ustilago maydis secretes a plethora of uncharacterized effector proteins and causes smut disease in maize. Among the effector genes that are up-regulated during the biotrophic growth in maize, we identified vp1 (virulence promoting 1), which has an expression that was up-regulated and maintained at a high level throughout the life cycle of the fungus. We characterized Vp1 by applying in silico analysis, reverse genetics, phenotypic assessment, microscopy, and protein localization and provided a fundamental understanding of the Vp1 protein in U. maydis. The reduction in fungal virulence and colonization in the vp1 mutant suggests the virulence-promoting function of Vp1. The deletion studies on the NLS (nuclear localization signal) sequence and the protein localization study revealed that the C-terminus of Vp1 is processed after secretion in plant apoplast and could localize to the plant nucleus. The Ustilago hordei ortholog UhVp1 lacks NLS localized in the plant cytoplasm, suggesting that the orthologs might have a distinct subcellular localization. Further complementation studies of the Vp1 orthologs in related smut fungi revealed that none of them could complement the virulence function of U. maydis Vp1, suggesting that UmVp1 could acquire a specialized function via sequence divergence.

Agrobacterium VirE2 Protein Modulates Plant Gene Expression and Mediates Transformation From Its Location Outside the Nucleus.

Agrobacterium effector protein VirE2 is important for plant transformation. VirE2 likely coats transferred DNA (T-DNA) in the plant cell and protects it from degradation. VirE2 localizes to the plant cytoplasm and interacts with several host proteins. Plant-expressed VirE2 can complement a virE2 mutant Agrobacterium strain to support transformation. We investigated whether VirE2 could facilitate transformation from a nuclear location by affixing to it a strong nuclear localization signal (NLS) sequence. Only cytoplasmic-, but not nuclear-localized, VirE2 could stimulate transformation. To investigate the ways VirE2 supports transformation, we generated transgenic Arabidopsis plants containing a virE2 gene under the control of an inducible promoter and performed RNA-seq and proteomic analyses before and after induction. Some differentially expressed plant genes were previously known to facilitate transformation. Knockout mutant lines of some other VirE2 differentially expressed genes showed altered transformation phenotypes. Levels of some proteins known to be important for transformation increased in response to VirE2 induction, but prior to or without induction of their corresponding mRNAs. Overexpression of some other genes whose proteins increased after VirE2 induction resulted in increased transformation susceptibility. We conclude that cytoplasmically localized VirE2 modulates both plant RNA and protein levels to facilitate transformation.