•Repeat-containing secreted effectors evolve rapidly.•Repeat-containing effectors allow fast adaptation to changing environments.•Repeat-containing effectors promote successful host colonization ...through diverse functions.•Repeat-containing effectors can protect microbes or function inside host cells.
Pathogenic and symbiotic filamentous microbes secrete effectors which suppress host immune responses and promote a successful colonization. Pathogen effectors are engaged in the arms race with their hosts and because of this they are subject to intense evolutionary pressure. Effectors particularly prone to rapid evolution display repeat-containing domains which can easily expand or contract and accumulate point mutations without altering their original function. In this review we address the diversity of function in such repeat-containing effectors, focus on new findings and point out avenues for future work.
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.
Plant pathogenic fungi colonizing living plant tissue secrete a cocktail of effector proteins to suppress plant immunity and reprogramme host cells. Although many of these effectors function inside ...host cells, delivery systems used by pathogenic bacteria to translocate effectors into host cells have not been detected in fungi. Here, we show that five unrelated effectors and two membrane proteins from Ustilago maydis, a biotrophic fungus causing smut disease in corn, form a stable protein complex. All seven genes appear co-regulated and are only expressed during colonization. Single mutants arrest in the epidermal layer, fail to suppress host defence responses and fail to induce non-host resistance, two reactions that likely depend on translocated effectors. The complex is anchored in the fungal membrane, protrudes into host cells and likely contacts channel-forming plant plasma membrane proteins. Constitutive expression of all seven complex members resulted in a surface-exposed form in cultured U. maydis cells. As orthologues of the complex-forming proteins are conserved in smut fungi, the complex may become an interesting fungicide target.
The type VI secretion system (T6SS) is a widespread protein secretion system found in many Gram-negative bacteria. T6SSs are highly regulated by various regulatory systems at multiple levels, ...including post-translational regulation via threonine (Thr) phosphorylation. The Ser/Thr protein kinase PpkA is responsible for this Thr phosphorylation regulation, and the forkhead-associated (FHA) domain-containing Fha-family protein is the sole T6SS phosphorylation substrate identified to date. Here we discovered that TssL, the T6SS inner-membrane core component, is phosphorylated and the phosphorylated TssL (p-TssL) activates type VI subassembly and secretion in a plant pathogenic bacterium, Agrobacterium tumefaciens. Combining genetic and biochemical approaches, we demonstrate that TssL is phosphorylated at Thr 14 in a PpkA-dependent manner. Further analysis revealed that the PpkA kinase activity is responsible for the Thr 14 phosphorylation, which is critical for the secretion of the T6SS hallmark protein Hcp and the putative toxin effector Atu4347. TssL phosphorylation is not required for the formation of the TssM-TssL inner-membrane complex but is critical for TssM conformational change and binding to Hcp and Atu4347. Importantly, Fha specifically interacts with phosphothreonine of TssL via its pThr-binding motif in vivo and in vitro and this interaction is crucial for TssL interaction with Hcp and Atu4347 and activation of type VI secretion. In contrast, pThr-binding ability of Fha is dispensable for TssM structural transition. In conclusion, we discover a novel Thr phosphorylation event, in which PpkA phosphorylates TssL to activate type VI secretion via its direct binding to Fha in A. tumefaciens. A model depicting an ordered TssL phosphorylation-induced T6SS assembly pathway is proposed.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The type VI secretion system (T6SS) is a widespread protein secretion system found in many Gram-negative bacteria. T6SSs are highly regulated by various regulatory systems at multiple levels, ...including post-translational regulation via threonine (Thr) phosphorylation. The Ser/Thr protein kinase PpkA is responsible for this Thr phosphorylation regulation, and the forkhead-associated (FHA) domain-containing Fha-family protein is the sole T6SS phosphorylation substrate identified to date. Here we discovered that TssL, the T6SS inner-membrane core component, is phosphorylated and the phosphorylated TssL (p-TssL) activates type VI subassembly and secretion in a plant pathogenic bacterium, Agrobacterium tumefaciens. Combining genetic and biochemical approaches, we demonstrate that TssL is phosphorylated at Thr 14 in a PpkA-dependent manner. Further analysis revealed that the PpkA kinase activity is responsible for the Thr 14 phosphorylation, which is critical for the secretion of the T6SS hallmark protein Hcp and the putative toxin effector Atu4347. TssL phosphorylation is not required for the formation of the TssM-TssL inner-membrane complex but is critical for TssM conformational change and binding to Hcp and Atu4347. Importantly, Fha specifically interacts with phosphothreonine of TssL via its pThr-binding motif in vivo and in vitro and this interaction is crucial for TssL interaction with Hcp and Atu4347 and activation of type VI secretion. In contrast, pThr-binding ability of Fha is dispensable for TssM structural transition. In conclusion, we discover a novel Thr phosphorylation event, in which PpkA phosphorylates TssL to activate type VI secretion via its direct binding to Fha in A. tumefaciens. A model depicting an ordered TssL phosphorylation-induced T6SS assembly pathway is proposed.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
An intracellular multiplication F (IcmF) family protein is a conserved component of a newly identified type VI secretion system (T6SS) encoded in many animal and plant-associated Proteobacteria. We ...have previously identified ImpL..., an IcmF family protein that is required for the secretion of the T6SS substrate hemolysin-coregulated protein (Hcp) from the plant-pathogenic bacterium Agrobacterium tumefaciens. In this study, we characterized the topology of ImpL... and the importance of its nucleotide-binding Walker A motif involved in Hcp secretion from A. tumefaciens. A combination of β-lactamase-green fluorescent protein fusion and biochemical fractionation analyses revealed that ImpL... is an integral polytopic inner membrane protein comprising three transmembrane domains bordered by an N-terminal domain facing the cytoplasm and a C-terminal domain exposed to the periplasm. impL... mutants with substitutions or deletions in the Walker A motif failed to complement the impL... deletion mutant for Hcp secretion, which provided evidence that ImpL... may bind and/or hydrolyze nucleoside triphosphates to mediate T6SS machine assembly and/or substrate secretion. Protein-protein interaction and protein stability analyses indicated that there is a physical interaction between ImpL... and another essential T6SS component, ImpK... Topology and biochemical fractionation analyses suggested that ImpK... is an integral bitopic inner membrane protein with an N-terminal domain facing the cytoplasm and a C-terminal OmpA-like domain exposed to the periplasm. Further comprehensive yeast two-hybrid assays dissecting ImpL...-ImpK... interaction domains suggested that ImpL... interacts with ImpK... via the N-terminal cytoplasmic domains of the proteins. In conclusion, ImpL... interacts with ImpK..., and its Walker A motif is required for its function in mediation of Hcp secretion from A. tumefaciens. (ProQuest: ... denotes formulae/symbols omitted.)
ABSTRACT
An intracellular multiplication F (IcmF) family protein is a conserved component of a newly identified type VI secretion system (T6SS) encoded in many animal and plant-associated
...Proteobacteria
. We have previously identified ImpL
M
, an IcmF family protein that is required for the secretion of the T6SS substrate hemolysin-coregulated protein (Hcp) from the plant-pathogenic bacterium
Agrobacterium tumefaciens
. In this study, we characterized the topology of ImpL
M
and the importance of its nucleotide-binding Walker A motif involved in Hcp secretion from
A. tumefaciens
. A combination of β-lactamase-green fluorescent protein fusion and biochemical fractionation analyses revealed that ImpL
M
is an integral polytopic inner membrane protein comprising three transmembrane domains bordered by an N-terminal domain facing the cytoplasm and a C-terminal domain exposed to the periplasm.
impL
M
mutants with substitutions or deletions in the Walker A motif failed to complement the
impL
M
deletion mutant for Hcp secretion, which provided evidence that ImpL
M
may bind and/or hydrolyze nucleoside triphosphates to mediate T6SS machine assembly and/or substrate secretion. Protein-protein interaction and protein stability analyses indicated that there is a physical interaction between ImpL
M
and another essential T6SS component, ImpK
L
. Topology and biochemical fractionation analyses suggested that ImpK
L
is an integral bitopic inner membrane protein with an N-terminal domain facing the cytoplasm and a C-terminal OmpA-like domain exposed to the periplasm. Further comprehensive yeast two-hybrid assays dissecting ImpL
M
-ImpK
L
interaction domains suggested that ImpL
M
interacts with ImpK
L
via the N-terminal cytoplasmic domains of the proteins. In conclusion, ImpL
M
interacts with ImpK
L
, and its Walker A motif is required for its function in mediation of Hcp secretion from
A. tumefaciens
.
The type VI secretion system (T6SS) is widely distributed in pathogenic Proteobacteria. Sequence and structural analysis of T6SS reveals a resemblance to the T4 bacteriophage tail, in which an outer ...sheath structure contracts an internal tube for injecting nucleic acid into bacterial cells. However, the molecular details of how this phage tail-like T6SS structure is assembled in vivo and executed for exoprotein or effector secretion remain largely unknown. Here, we used a systematic approach to identify T6SS machinery and secreted components and investigate the interaction among the putative sheath and tube components of Agrobacterium tumefaciens. We showed that 14 T6SS components play essential roles in the secretion of the T6SS hallmark exoprotein Hcp. In addition, we discovered a novel T6SS exoprotein, Atu4347, that is dispensable for Hcp secretion. Interestingly, Atu4347 and the putative tube components, Hcp and VgrG, are mainly localized in the cytoplasm but also detected on the bacterial surface. Atu4342 (TssB) and Atu4341 (TssC41) interact with and stabilize each other, which suggests that they are functional orthologs of the sheath components TssB (VipA) and TssC (VipB), respectively. Importantly, TssB interacts directly with the three exoproteins (Hcp, VgrG, and Atu4347), in which Hcp also interacts directly with VgrG-1 on co-purification from Escherichia coli. Further co-immunoprecipitation and pulldown assays revealed these subcomplex(es) in A. tumefaciens and thereby support T6SS functioning as a contractile phage tail-like structure.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
A normalized embryoid cDNA library (EON) was constructed based on
reassociation kinetics reaction. Results from dot blot hybridization
and sequencing of EON cDNA clones clearly indicated that the
...normalization process reduced the frequency of high abundance
transcripts and increased the frequency of low abundance gene
transcripts. A total of 553 non-redundant expressed sequence tags
(ESTs) were identified, 325 of these were not observed in the standard
oil palm cDNA libraries sequenced previously. A total of 10 EON cDNA
clones were chosen for expression profiling across samples from
different stages of the tissue culture process. Two of the genes
exhibited promising expression patterns for predicting the embryogenic
potential in callus. Some of these genes were also differentially
expressed in the various tissues of oil palm. This study showed that
normalization of the existing embryoid library improved the chances of
identifying transcripts not captured in the standard libraries, some of
which could be associated with embryogenesis. This collection of ESTs
is particularly well suited for use as candidate genes for development
of an oil palm DNA chip, which can be used to obtain a more
comprehensive view of the molecular mechanism associated with oil palm
tissue culture.
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