Data_Sheet_1_NLRexpress—A bundle of machine learning motif predictors—Reveals motif stability underlying plant Nod-like receptors diversity.PDF

Examination of a collection of over 80,000 Plant Nod-like receptors (NLRs) revealed an overwhelming sequence diversity underlying functional specificity of pathogen detection, signaling and cooperativity. The NLR canonical building blocks—CC/TIR/RPW8, NBS and LRR—contain, however, a number of conserved sequence motifs showing a significant degree of invariance amongst different NLR groups. To identify these motifs we developed NLRexpress—a bundle of 17 machine learning (ML)-based predictors, able to swiftly and precisely detect CC, TIR, NBS, and LRR motifs while minimizing computing time without accuracy losses—aimed as an instrument scalable for screening overall proteomes, transcriptomes or genomes for identifying integral NLRs and discriminating them against incomplete sequences lacking key motifs. These predictors were further used to screen a subset of ∼34,000 regular plant NLR sequences. Motifs were analyzed using unsupervised ML techniques to assess the structural correlations hidden underneath pattern variabilities. Both the NB-ARC switch domain which admittedly is the most conserved region of NLRs and the highly diverse LRR domain with its vastly variable lengths and repeat irregularities—show well-defined relations between motif subclasses, highlighting the importance of structural invariance in shaping NLR sequence diversity. The online NLRexpress webserver can be accessed at https://nlrexpress.biochim.ro.</p

Analysis of expression of a stable monomeric form of human IL-10.

<p>A stable monomeric form of human IL-10 (hIL-10^mono) does not granulate and yield increases 30-fold. (A) Three cartoons illustrating the human IL-10 (I) dimer, (II) monomer and (III) stable monomer structure, as well as a schematic representation of the human (h) IL-10 alpha helices A–F. Helices are represented by ovals, whereby a fragment of the amino acid sequence and the location of insertion of the small GS-linker is indicated. (B) Whole mount confocal microscopy output of GFP fused C-terminally to hIL-10^mono including native signal peptide (SP). (C) Western blot analysis under non-reducing conditions of plant produced hIL-10 and hIL-10^mono. As controls, empty vector (EV) and 50 ng recombinant (r) <i>E. coli</i> produced hL-10 were used. A molecular weight marker is indicated in kDa. (D) Yield of hIL-10 and hIL-10^mono in crude extracts 2 to 5 days post infiltration as determined by ELISA (<i>n</i> = 3, error bars indicate standard error). Average yield of hIL-10^mono was significantly higher compared to hIL-10.</p

Expression data of human and mouse IL-10 in transiently transformed <i>Nicotiana benthamiana</i> leaves.

<p>Use of the thk-tag gives an increasing boost in yield for both human and mouse IL-10 from 2 days post infiltration (dpi). Strikingly, mouse IL-10 yield was significantly higher compared to human IL-10, regardless of ER-retention. Differences in yield could not be explained by differences in mRNA transcript levels. (A) Schematic representation of expression cassettes and vector used. Expressed genes include the native coding sequence of the human (h) or mouse (m) IL-10 gene including signal peptide for secretion (SP) with or without a 3′ tag coding for a thrombin cleavage site, a 6xHis-tag and the ER retention sequence KDEL (thk). All expression cassettes include the 35S promoter of the Cauliflower mosaic virus with duplicated enhancer (d35S), 5′ leader sequence of the Alfalfa mosaic virus RNA 4 (AlMV) and <i>Agrobacterium tumefaciens</i> nopaline synthase transcription terminator (Tnos). (B) Relative transcript levels of IL-10 versus actin as determined by Q-PCR on 2 and 3 dpi (<i>n</i> = 3, error bars indicate standard error). (C/D) Human and mouse IL-10 yield in crude extracts (1 to 6 dpi) in µg per mg total soluble protein (TSP) as determined by ELISA (<i>n</i> = 3, error bars indicate standard error).</p

Analysis of the effect of <i>N</i>-glycosylation at Asn29 on granulation.

<p>Glycosylation of IL-10 plays a role in preventing granulation. (A/B) Whole mount confocal microscopy output of leaves expressing GFP fused C-terminally to human (h) and mouse (m) IL-10 including native signal peptide (SP) and with introduced (S29N) or removed (N29S) glycosylation site, respectively. (C/D) Western blot analysis under reducing conditions of plant produced (p) hIL-10 and mIL-10 with and without glycosylation site. As controls, empty vector (EV) and 50 ng recombinant (r) <i>E. coli</i> produced hL-10 and mIL-10 were used. A molecular weight marker is indicated in kDa. (E/F) Yield of hIL-10 and mIL-10 with and without glycosylation site in crude extracts 2 to 5 days post infiltration (dpi) as determined by ELISA (<i>n</i> = 4, error bars indicate standard error).</p

Biological activity of human and mouse IL-10 variants on human and mouse macrophages.

<p>Plant produced (p) and recombinant (r) <i>E. coli</i> produced human (h) or mouse (m) IL-10 were calibrated to contain the same amount of IL-10 as well as total soluble protein by using the empty vector control. Human (THP-1) and mouse (RAW264.7) macrophages were then pretreated with 10 ng/ml hIL-10 or mIL-10 for 20 min and subsequently stimulated with 1 µg/ml <i>E. coli</i> lipopolysaccharide. Tumor Necrosis Factor-alpha (TNF-α) expression was determined by ELISA and IL-10 activity is indicated as the percentage of inhibition of TNF-α expression as compared to the empty vector control (<i>n</i> = 3, error bars indicate standard error).</p

Whole mount confocal microscopy output of leaves expressing human or mouse IL-10 fused to GFP.

<p>Highly mobile globular granules of up to 5 µm in size were observed traveling along cytoplasmic and/or ER strands for SP-hIL-10-GFP only. (A) GFP preceded by the <i>Arabidopsis thaliana</i> chitinase signal peptide for secretion (SP-GFP). (B/C) The native open reading frame of human (h) and mouse (m) IL-10 including the native signal peptide (SP) with GFP fused C-terminally.</p

Oligonucleotides used for construct re-amplification, mutagenesis and insertion.

<p>Native sequences in capitals, added/mutated sequences in small and restriction sites are underlined. h; human, IL-10; interleukin-10, MCS; multiple cloning site, m; mouse, Nb; <i>Nicotiana</i> benthamiana, SP; signal peptide for secretion, thk; thrombin-6xHIS-KDEL tag.</p

Image_2_The Globodera pallida SPRYSEC Effector GpSPRY-414-2 That Suppresses Plant Defenses Targets a Regulatory Component of the Dynamic Microtubule Network.JPEG

<p>The white potato cyst nematode, Globodera pallida, is an obligate biotrophic pathogen of a limited number of Solanaceous plants. Like other plant pathogens, G. pallida deploys effectors into its host that manipulate the plant to the benefit of the nematode. Genome analysis has led to the identification of large numbers of candidate effectors from this nematode, including the cyst nematode-specific SPRYSEC proteins. These are a secreted subset of a hugely expanded gene family encoding SPRY domain-containing proteins, many of which remain to be characterized. We investigated the function of one of these SPRYSEC effector candidates, GpSPRY-414-2. Expression of the gene encoding GpSPRY-414-2 is restricted to the dorsal pharyngeal gland cell and reducing its expression in G. pallida infective second stage juveniles using RNA interference causes a reduction in parasitic success on potato. Transient expression assays in Nicotiana benthamiana indicated that GpSPRY-414-2 disrupts plant defenses. It specifically suppresses effector-triggered immunity (ETI) induced by co-expression of the Gpa2 resistance gene and its cognate avirulence factor RBP-1. It also causes a reduction in the production of reactive oxygen species triggered by exposure of plants to the bacterial flagellin epitope flg22. Yeast two-hybrid screening identified a potato cytoplasmic linker protein (CLIP)-associated protein (StCLASP) as a host target of GpSPRY-414-2. The two proteins co-localize in planta at the microtubules. CLASPs are members of a conserved class of microtubule-associated proteins that contribute to microtubule stability and growth. However, disruption of the microtubule network does not prevent suppression of ETI by GpSPRY-414-2 nor the interaction of the effector with its host target. Besides, GpSPRY-414-2 stabilizes its target while effector dimerization and the formation of high molecular weight protein complexes including GpSPRY-414-2 are prompted in the presence of the StCLASP. These data indicate that the nematode effector GpSPRY-414-2 targets the microtubules to facilitate infection.</p

Image_5_The Globodera pallida SPRYSEC Effector GpSPRY-414-2 That Suppresses Plant Defenses Targets a Regulatory Component of the Dynamic Microtubule Network.JPEG

<p>The white potato cyst nematode, Globodera pallida, is an obligate biotrophic pathogen of a limited number of Solanaceous plants. Like other plant pathogens, G. pallida deploys effectors into its host that manipulate the plant to the benefit of the nematode. Genome analysis has led to the identification of large numbers of candidate effectors from this nematode, including the cyst nematode-specific SPRYSEC proteins. These are a secreted subset of a hugely expanded gene family encoding SPRY domain-containing proteins, many of which remain to be characterized. We investigated the function of one of these SPRYSEC effector candidates, GpSPRY-414-2. Expression of the gene encoding GpSPRY-414-2 is restricted to the dorsal pharyngeal gland cell and reducing its expression in G. pallida infective second stage juveniles using RNA interference causes a reduction in parasitic success on potato. Transient expression assays in Nicotiana benthamiana indicated that GpSPRY-414-2 disrupts plant defenses. It specifically suppresses effector-triggered immunity (ETI) induced by co-expression of the Gpa2 resistance gene and its cognate avirulence factor RBP-1. It also causes a reduction in the production of reactive oxygen species triggered by exposure of plants to the bacterial flagellin epitope flg22. Yeast two-hybrid screening identified a potato cytoplasmic linker protein (CLIP)-associated protein (StCLASP) as a host target of GpSPRY-414-2. The two proteins co-localize in planta at the microtubules. CLASPs are members of a conserved class of microtubule-associated proteins that contribute to microtubule stability and growth. However, disruption of the microtubule network does not prevent suppression of ETI by GpSPRY-414-2 nor the interaction of the effector with its host target. Besides, GpSPRY-414-2 stabilizes its target while effector dimerization and the formation of high molecular weight protein complexes including GpSPRY-414-2 are prompted in the presence of the StCLASP. These data indicate that the nematode effector GpSPRY-414-2 targets the microtubules to facilitate infection.</p

Image_4_The Globodera pallida SPRYSEC Effector GpSPRY-414-2 That Suppresses Plant Defenses Targets a Regulatory Component of the Dynamic Microtubule Network.JPEG

<p>The white potato cyst nematode, Globodera pallida, is an obligate biotrophic pathogen of a limited number of Solanaceous plants. Like other plant pathogens, G. pallida deploys effectors into its host that manipulate the plant to the benefit of the nematode. Genome analysis has led to the identification of large numbers of candidate effectors from this nematode, including the cyst nematode-specific SPRYSEC proteins. These are a secreted subset of a hugely expanded gene family encoding SPRY domain-containing proteins, many of which remain to be characterized. We investigated the function of one of these SPRYSEC effector candidates, GpSPRY-414-2. Expression of the gene encoding GpSPRY-414-2 is restricted to the dorsal pharyngeal gland cell and reducing its expression in G. pallida infective second stage juveniles using RNA interference causes a reduction in parasitic success on potato. Transient expression assays in Nicotiana benthamiana indicated that GpSPRY-414-2 disrupts plant defenses. It specifically suppresses effector-triggered immunity (ETI) induced by co-expression of the Gpa2 resistance gene and its cognate avirulence factor RBP-1. It also causes a reduction in the production of reactive oxygen species triggered by exposure of plants to the bacterial flagellin epitope flg22. Yeast two-hybrid screening identified a potato cytoplasmic linker protein (CLIP)-associated protein (StCLASP) as a host target of GpSPRY-414-2. The two proteins co-localize in planta at the microtubules. CLASPs are members of a conserved class of microtubule-associated proteins that contribute to microtubule stability and growth. However, disruption of the microtubule network does not prevent suppression of ETI by GpSPRY-414-2 nor the interaction of the effector with its host target. Besides, GpSPRY-414-2 stabilizes its target while effector dimerization and the formation of high molecular weight protein complexes including GpSPRY-414-2 are prompted in the presence of the StCLASP. These data indicate that the nematode effector GpSPRY-414-2 targets the microtubules to facilitate infection.</p