Infection assays in Arabidopsis reveal candidate effectors from the poplar rust fungus that promote susceptibility to bacteria and oomycete pathogens

Fungi of the Pucciniales order cause rust diseases which, altogether, affect thousands of plant species worldwide and pose a major threat to several crops. How rust effectors—virulence proteins delivered into infected tissues to modulate host functions— contribute to pathogen virulence remains poorly understood. Melampsora larici-populina is a devastating and widespread rust pathogen of poplar, and its genome encodes 1184 identified small secreted proteins that could potentially act as effectors. Here, following specific criteria, we selected 16 candidate effector proteins and characterized their virulence activities and subcellular localizations in the leaf cells of Arabidopsis thaliana. Infection assays using bacterial (Pseudomonas syringae) and oomycete (Hyaloperonospora arabidopsidis) pathogens revealed subsets of candidate effectors that enhanced or decreased pathogen leaf colonization. Confocal imaging of green fluorescent protein-tagged candidate effectors constitutively expressed in stable transgenic plants revealed that some protein fusions specifically accumulate in nuclei, chloroplasts, plasmodesmata and punctate cytosolic structures. Altogether, our analysis suggests that rust fungal candidate effectors target distinct cellular components in host cells to promote parasitic growth. © 2016 BSPP AND JOHN WILEY & SONS LTD

The V86M mutation in HIV-1 capsid confers resistance to TRIM5α by abrogation of cyclophilin A-dependent restriction and enhancement of viral nuclear import.

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.BACKGROUND: HIV-1 is inhibited early after entry into cells expressing some simian orthologues of the tripartite motif protein family member TRIM5α. Mutants of the human orthologue (TRIM5αhu) can also provide protection against HIV-1. The host protein cyclophilin A (CypA) binds incoming HIV-1 capsid (CA) proteins and enhances early stages of HIV-1 replication by unknown mechanisms. On the other hand, the CA-CypA interaction is known to increase HIV-1 susceptibility to restriction by TRIM5α. Previously, the mutation V86M in the CypA-binding loop of HIV-1 CA was found to be selected upon serial passaging of HIV-1 in cells expressing Rhesus macaque TRIM5α (TRIM5αrh). The objectives of this study were (i) to analyze whether V86M CA allows HIV-1 to escape mutants of TRIM5αhu, and (ii) to characterize the role of CypA in the resistance to TRIM5α conferred by V86M. RESULTS: We find that in single-cycle HIV-1 vector transduction experiments, V86M confers partial resistance against R332G-R335G TRIM5αhu and other TRIM5αhu variable 1 region mutants previously isolated in mutagenic screens. However, V86M HIV-1 does not seem to be resistant to R332G-R335G TRIM5αhu in a spreading infection context. Strikingly, restriction of V86M HIV-1 vectors by TRIM5αhu mutants is mostly insensitive to the presence of CypA in infected cells. NMR experiments reveal that V86M alters CypA interactions with, and isomerisation of CA. On the other hand, V86M does not affect the CypA-mediated enhancement of HIV-1 replication in permissive human cells. Finally, qPCR experiments show that V86M increases HIV-1 transport to the nucleus of cells expressing restrictive TRIM5α. CONCLUSIONS: Our study shows that V86M de-couples the two functions associated with CA-CypA binding, i.e. the enhancement of restriction by TRIM5α and the enhancement of HIV-1 replication in permissive human cells. V86M enhances the early stages of HIV-1 replication in restrictive cells by improving nuclear import. In summary, our data suggest that HIV-1 escapes restriction by TRIM5α through the selective disruption of CypA-dependent, TRIM5α-mediated inhibition of nuclear import. However, V86M does not seem to relieve restriction of a spreading HIV-1 infection by TRIM5αhu mutants, underscoring context-specific restriction mechanisms

Étude des protéines angiomotine et angiomotine-like1

La famille des motines, composée d'angiomotine, d'angiomotin-like 1 et d'angiomotin-like 2, est impliquée dans la migration cellulaire, les jonctions cellule-cellule et Tangiogenèse. Comme la formation de nouveaux vaisseaux sanguins est un élément important de la pathologie de la polyarthrite rhumatoïde et qu'il est connu qu'angiomotine accroît Tangiogenèse, son expression a été étudiée dans des cellules endothéliales soumises à des conditions pro-inflammatoires ressemblant à la polyarthrite rhumatoïde. Cependant, aucune variation notable n'a été observée. Un nouveau partenaire protéique d'angiomotine, angiomotin-like 1, a été identifié par co-immunoprécipitation. Il a été observé que la surexpression d'angiomotine like 1 affecte la morphologie du cytosquelette d'actine. De plus, angiomotine-like 1 colocalise au front de migration avec ARNO, une protéine ayant entre autres fonctions d'augmenter la formation de lamellipodes et la motilité cellulaire. Cependant, contrairement à angiomotine, angiomotin-like 1 n'a pas d'effet sur la migration cellulaire aléatoire. Ces travaux ont permis de mieux définir différents aspects de la biologie de deux membres de la famille des motines, soit angiomotine et angiomotin-like 1

Host-Specific and Homologous Pairs of Melampsora larici-populina Effectors Unveil Novel Nicotiana benthamiana Stromule Induction Factors

The poplar rust fungus Melampsora larici-populina is part of one of the most devastating group of fungi (Pucciniales) and causes important economic losses to the poplar industry. Because M. larici-populina is a heteroecious obligate biotroph, its spread depends on its ability to carry out its reproductive cycle through larch and then poplar parasitism. Genomic approaches have identified more than 1,000 candidate secreted effector proteins (CSEPs) from the predicted secretome of M. larici-populina that are potentially implicated in the infection process. In this study, we selected CSEP pairs (and one triplet) among CSEP gene families that share high sequence homology but display specific gene expression profiles among the two distinct hosts. We determined their subcellular localization by confocal microscopy through expression in the heterologous plant system Nicotiana benthamiana. Five out of nine showed partial or complete chloroplastic localization. We also screened for potential protein interactors from larch and poplar by yeast two-hybrid assays. One pair of CSEPs and the triplet shared common interactors, whereas the members of the two other pairs did not have common targets from either host. Finally, stromule induction quantification revealed that two pairs and the triplet of CSEPs induced stromules when transiently expressed in N. benthamiana. The use of N. benthamiana eds1 and nrg1 knockout lines showed that CSEPs can induce stromules through an eds1-independent mechanism. However, CSEP homologs shared the same impact on stromule induction and contributed to discovering a new stromule induction cascade that can be partially and/or fully independent of eds1. [Graphic: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license

A putative SUMO interacting motif in the B30.2/SPRY domain of rhesus macaque TRIM5α important for NF-κB/AP-1 signaling and HIV-1 restriction

TRIM5α from the rhesus macaque (TRIM5αRh) is a restriction factor that shows strong activity against HIV-1. TRIM5αRh binds specifically to HIV-1 capsid (CA) through its B30.2/PRYSPRY domain shortly after entry of the virus into the cytoplasm. Recently, three putative SUMO interacting motifs (SIMs) have been identified in the PRYSPRY domain of human and macaque TRIM5α. However, structural modeling of this domain suggested that two of them were buried in the hydrophobic core of the protein, implying that interaction with SUMO was implausible, while the third one was not relevant to restriction. In light of these results, we re-analyzed the TRIM5αRh PRYSPRY sequence and identified an additional putative SIM (435VIIC438) which we named SIM4. This motif is exposed at the surface of the PRYSPRY domain, allowing potential interactions with SUMO or SUMOylated proteins. Introducing a double mutation in SIM4 (V435K, I436K) did not alter stability, unlike mutations in SIM1. SIM4-mutated TRIM5αRh failed to bind HIV-1CA and lost the ability to restrict this virus. Accordingly, SIM4 undergoes significant variation among primates and substituting this motif with naturally occurring SIM4 variants affected HIV-1 restriction by TRIM5αRh, suggesting a direct role in capsid recognition. Interestingly, SIM4-mutated TRIM5αRh also failed to activate NF-κB and AP-1-mediated transcription. Although there is no direct evidence that SIM4 is involved in direct interaction with SUMO or a SUMOylated protein, mutating this motif strongly reduced co-localization of TRIM5αRh with SUMO-1 and with PML, a SUMOylated nuclear protein. In conclusion, this new putative SIM is crucial for both direct interaction with incoming capsids and for NF-κB/AP-1 signaling. We speculate that the latter function is mediated by interactions of SIM4 with a SUMOylated protein involved in the NF-κB/AP-1 signaling pathways

HIV-1 capsids from B27/B57+ elite controllers escape Mx2 but are targeted by TRIM5α, leading to the induction of an antiviral state.

Elite controllers (ECs) are a rare subset of HIV-1 slow progressors characterized by prolonged viremia suppression. HLA alleles B27 and B57 promote the cytotoxic T lymphocyte (CTL)-mediated depletion of infected cells in ECs, leading to the emergence of escape mutations in the viral capsid (CA). Whether those mutations modulate CA detection by innate sensors and effectors is poorly known. Here, we investigated the targeting of CA from B27/B57+ individuals by cytosolic antiviral factors Mx2 and TRIM5α. Toward that aim, we constructed chimeric HIV-1 vectors using CA isolated from B27/B57+ or control subjects. HIV-1 vectors containing B27/B57+-specific CA had increased sensitivity to TRIM5α but not to Mx2. Following exposure to those vectors, cells showed increased resistance against both TRIM5α-sensitive and -insensitive HIV-1 strains. Induction of the antiviral state did not require productive infection by the TRIM5α-sensitive virus, as shown using chemically inactivated virions. Depletion experiments revealed that TAK1 and Ubc13 were essential to the TRIM5α-dependent antiviral state. Accordingly, induction of the antiviral state was accompanied by the activation of NF-κB and AP-1 in THP-1 cells. Secretion of IFN-I was involved in the antiviral state in THP-1 cells, as shown using a receptor blocking antibody. This work identifies innate activation pathways that are likely to play a role in the natural resistance to HIV-1 progression in ECs

Pre-screening of isolated clones by specific PCR.

<p>Following 20 days of growth, 161 isolated HEK293T clones were screened for HDR-edited TRIM5 gene by PCR using a primer specific for the mutated <i>TRIM5</i>. 14 clones that passed this pre-screen step are indicated by their names. MWM, molecular weight marker.</p

Design of the gRNA and donor ssODN for the HDR-mediated editing of <i>TRIM5</i>.

<p>(A) <i>TRIM5</i> localization on chromosome 11 (top), and Arg332-Arg335 localization in exon 8 of the gene (bottom). (B) Top panel: position of the three gRNAs (gRNA1, 9 and 19) designed to target the Arg332-Arg335 region. The two arginine codons are underlined and in bold. Bottom panel: Surveyor assay following the transfection of HEK293T cells with CRISPR-Cas9 plasmids expressing one of the three gRNAs. WT DNA from untransfected cells was used as a control. (C) HDR donor DNA mutagenesis strategy. 8 substitutions were present, including three nonsilent substitutions to mutate Arg332 and Arg335 into Gly (green), one silent mutation to disrupt the PAM sequence (pink), and four silent mutations in the sequence targeted by gRNA1 (orange). The HaeIII restriction site created as a result of one of the silent substitutions is indicated, as is the position of the primer used in specific PCR screening.</p

Identification of HDR-edited clones.

<p>(A) Mutation-specific PCR was performed on 13 clones showing a positive signal in the pre-screen. Untransfected HEK293T cells were used as a control. M, molecular weight marker. (B) Non-specific PCR of the targeted region followed by HaeIII digestion. The expected sizes of the digested PCR products are shown on the right. The full-length gels are available on the FigShare public repository (see “Availability of data” section).</p

Deep sequencing analysis of <i>TRIM5</i> editing in 10 screened clones.

<p>The ~200-nt HDR-targeted <i>TRIM5</i> region was amplified by PCR and the PCR products were then analyzed by Illumina MiSeq sequencing. The alignment shown includes the targeted locus for each allele of the 10 clones, in comparison with the WT sequence and with the expected HDR-mutated sequence (top 2 lines). The codons for aa 332 and 335 are in bold. The Cas9 cleavage site on the WT sequence is shown at the top (arrow). Expected substitutions are shown in green (R332G/R335G), in orange (silent mutations in the gRNA target sequence) and in pink (silent mutation in the PAM). Duplications/insertions are underlined whereas deletions are represented by dashes. The star indicates the position of an unexpected substitution within a duplicated region in one allele of F2X. On the left is a table summarizing the results obtained for each clone: presence of the two therapeutic mutations R332G/R335G, proportion of <i>TRIM5</i> alleles modified by HDR and the proportion of the expected substitution mutations in the HDR-edited alleles. Note that only one clone (D11) has an allele containing all the desired mutations and that most of the non-HDR-edited alleles contain indels at the cleavage site.</p