Table_2_Subcellular Localization Screening of Colletotrichum higginsianum Effector Candidates Identifies Fungal Proteins Targeted to Plant Peroxisomes, Golgi Bodies, and Microtubules.PDF

<p>The genome of the hemibiotrophic anthracnose fungus, Colletotrichum higginsianum, encodes a large inventory of putative secreted effector proteins that are sequentially expressed at different stages of plant infection, namely appressorium-mediated penetration, biotrophy and necrotrophy. However, the destinations to which these proteins are addressed inside plant cells are unknown. In the present study, we selected 61 putative effector genes that are highly induced in appressoria and/or biotrophic hyphae. We then used Agrobacterium-mediated transformation to transiently express them as N-terminal fusions with fluorescent proteins in cells of Nicotiana benthamiana for imaging by confocal microscopy. Plant compartments labeled by the fusion proteins in N. benthamiana were validated by co-localization with specific organelle markers, by transient expression of the proteins in the true host plant, Arabidopsis thaliana, and by transmission electron microscopy-immunogold labeling. Among those proteins for which specific subcellular localizations could be verified, nine were imported into plant nuclei, three were imported into the matrix of peroxisomes, three decorated cortical microtubule arrays and one labeled Golgi stacks. Two peroxisome-targeted proteins harbored canonical C-terminal tripeptide signals for peroxisome import via the PTS1 (peroxisomal targeting signal 1) pathway, and we showed that these signals are essential for their peroxisome localization. Our findings provide valuable information about which host processes are potentially manipulated by this pathogen, and also reveal plant peroxisomes, microtubules, and Golgi as novel targets for fungal effectors.</p

Image_3_Subcellular Localization Screening of Colletotrichum higginsianum Effector Candidates Identifies Fungal Proteins Targeted to Plant Peroxisomes, Golgi Bodies, and Microtubules.PDF

<p>The genome of the hemibiotrophic anthracnose fungus, Colletotrichum higginsianum, encodes a large inventory of putative secreted effector proteins that are sequentially expressed at different stages of plant infection, namely appressorium-mediated penetration, biotrophy and necrotrophy. However, the destinations to which these proteins are addressed inside plant cells are unknown. In the present study, we selected 61 putative effector genes that are highly induced in appressoria and/or biotrophic hyphae. We then used Agrobacterium-mediated transformation to transiently express them as N-terminal fusions with fluorescent proteins in cells of Nicotiana benthamiana for imaging by confocal microscopy. Plant compartments labeled by the fusion proteins in N. benthamiana were validated by co-localization with specific organelle markers, by transient expression of the proteins in the true host plant, Arabidopsis thaliana, and by transmission electron microscopy-immunogold labeling. Among those proteins for which specific subcellular localizations could be verified, nine were imported into plant nuclei, three were imported into the matrix of peroxisomes, three decorated cortical microtubule arrays and one labeled Golgi stacks. Two peroxisome-targeted proteins harbored canonical C-terminal tripeptide signals for peroxisome import via the PTS1 (peroxisomal targeting signal 1) pathway, and we showed that these signals are essential for their peroxisome localization. Our findings provide valuable information about which host processes are potentially manipulated by this pathogen, and also reveal plant peroxisomes, microtubules, and Golgi as novel targets for fungal effectors.</p

Image_4_Subcellular Localization Screening of Colletotrichum higginsianum Effector Candidates Identifies Fungal Proteins Targeted to Plant Peroxisomes, Golgi Bodies, and Microtubules.PDF

<p>The genome of the hemibiotrophic anthracnose fungus, Colletotrichum higginsianum, encodes a large inventory of putative secreted effector proteins that are sequentially expressed at different stages of plant infection, namely appressorium-mediated penetration, biotrophy and necrotrophy. However, the destinations to which these proteins are addressed inside plant cells are unknown. In the present study, we selected 61 putative effector genes that are highly induced in appressoria and/or biotrophic hyphae. We then used Agrobacterium-mediated transformation to transiently express them as N-terminal fusions with fluorescent proteins in cells of Nicotiana benthamiana for imaging by confocal microscopy. Plant compartments labeled by the fusion proteins in N. benthamiana were validated by co-localization with specific organelle markers, by transient expression of the proteins in the true host plant, Arabidopsis thaliana, and by transmission electron microscopy-immunogold labeling. Among those proteins for which specific subcellular localizations could be verified, nine were imported into plant nuclei, three were imported into the matrix of peroxisomes, three decorated cortical microtubule arrays and one labeled Golgi stacks. Two peroxisome-targeted proteins harbored canonical C-terminal tripeptide signals for peroxisome import via the PTS1 (peroxisomal targeting signal 1) pathway, and we showed that these signals are essential for their peroxisome localization. Our findings provide valuable information about which host processes are potentially manipulated by this pathogen, and also reveal plant peroxisomes, microtubules, and Golgi as novel targets for fungal effectors.</p

Image_6_Subcellular Localization Screening of Colletotrichum higginsianum Effector Candidates Identifies Fungal Proteins Targeted to Plant Peroxisomes, Golgi Bodies, and Microtubules.PDF

<p>The genome of the hemibiotrophic anthracnose fungus, Colletotrichum higginsianum, encodes a large inventory of putative secreted effector proteins that are sequentially expressed at different stages of plant infection, namely appressorium-mediated penetration, biotrophy and necrotrophy. However, the destinations to which these proteins are addressed inside plant cells are unknown. In the present study, we selected 61 putative effector genes that are highly induced in appressoria and/or biotrophic hyphae. We then used Agrobacterium-mediated transformation to transiently express them as N-terminal fusions with fluorescent proteins in cells of Nicotiana benthamiana for imaging by confocal microscopy. Plant compartments labeled by the fusion proteins in N. benthamiana were validated by co-localization with specific organelle markers, by transient expression of the proteins in the true host plant, Arabidopsis thaliana, and by transmission electron microscopy-immunogold labeling. Among those proteins for which specific subcellular localizations could be verified, nine were imported into plant nuclei, three were imported into the matrix of peroxisomes, three decorated cortical microtubule arrays and one labeled Golgi stacks. Two peroxisome-targeted proteins harbored canonical C-terminal tripeptide signals for peroxisome import via the PTS1 (peroxisomal targeting signal 1) pathway, and we showed that these signals are essential for their peroxisome localization. Our findings provide valuable information about which host processes are potentially manipulated by this pathogen, and also reveal plant peroxisomes, microtubules, and Golgi as novel targets for fungal effectors.</p

Image_1_Subcellular Localization Screening of Colletotrichum higginsianum Effector Candidates Identifies Fungal Proteins Targeted to Plant Peroxisomes, Golgi Bodies, and Microtubules.PDF

<p>The genome of the hemibiotrophic anthracnose fungus, Colletotrichum higginsianum, encodes a large inventory of putative secreted effector proteins that are sequentially expressed at different stages of plant infection, namely appressorium-mediated penetration, biotrophy and necrotrophy. However, the destinations to which these proteins are addressed inside plant cells are unknown. In the present study, we selected 61 putative effector genes that are highly induced in appressoria and/or biotrophic hyphae. We then used Agrobacterium-mediated transformation to transiently express them as N-terminal fusions with fluorescent proteins in cells of Nicotiana benthamiana for imaging by confocal microscopy. Plant compartments labeled by the fusion proteins in N. benthamiana were validated by co-localization with specific organelle markers, by transient expression of the proteins in the true host plant, Arabidopsis thaliana, and by transmission electron microscopy-immunogold labeling. Among those proteins for which specific subcellular localizations could be verified, nine were imported into plant nuclei, three were imported into the matrix of peroxisomes, three decorated cortical microtubule arrays and one labeled Golgi stacks. Two peroxisome-targeted proteins harbored canonical C-terminal tripeptide signals for peroxisome import via the PTS1 (peroxisomal targeting signal 1) pathway, and we showed that these signals are essential for their peroxisome localization. Our findings provide valuable information about which host processes are potentially manipulated by this pathogen, and also reveal plant peroxisomes, microtubules, and Golgi as novel targets for fungal effectors.</p

Image_5_Subcellular Localization Screening of Colletotrichum higginsianum Effector Candidates Identifies Fungal Proteins Targeted to Plant Peroxisomes, Golgi Bodies, and Microtubules.PDF

<p>The genome of the hemibiotrophic anthracnose fungus, Colletotrichum higginsianum, encodes a large inventory of putative secreted effector proteins that are sequentially expressed at different stages of plant infection, namely appressorium-mediated penetration, biotrophy and necrotrophy. However, the destinations to which these proteins are addressed inside plant cells are unknown. In the present study, we selected 61 putative effector genes that are highly induced in appressoria and/or biotrophic hyphae. We then used Agrobacterium-mediated transformation to transiently express them as N-terminal fusions with fluorescent proteins in cells of Nicotiana benthamiana for imaging by confocal microscopy. Plant compartments labeled by the fusion proteins in N. benthamiana were validated by co-localization with specific organelle markers, by transient expression of the proteins in the true host plant, Arabidopsis thaliana, and by transmission electron microscopy-immunogold labeling. Among those proteins for which specific subcellular localizations could be verified, nine were imported into plant nuclei, three were imported into the matrix of peroxisomes, three decorated cortical microtubule arrays and one labeled Golgi stacks. Two peroxisome-targeted proteins harbored canonical C-terminal tripeptide signals for peroxisome import via the PTS1 (peroxisomal targeting signal 1) pathway, and we showed that these signals are essential for their peroxisome localization. Our findings provide valuable information about which host processes are potentially manipulated by this pathogen, and also reveal plant peroxisomes, microtubules, and Golgi as novel targets for fungal effectors.</p

Image_2_Subcellular Localization Screening of Colletotrichum higginsianum Effector Candidates Identifies Fungal Proteins Targeted to Plant Peroxisomes, Golgi Bodies, and Microtubules.PDF

<p>The genome of the hemibiotrophic anthracnose fungus, Colletotrichum higginsianum, encodes a large inventory of putative secreted effector proteins that are sequentially expressed at different stages of plant infection, namely appressorium-mediated penetration, biotrophy and necrotrophy. However, the destinations to which these proteins are addressed inside plant cells are unknown. In the present study, we selected 61 putative effector genes that are highly induced in appressoria and/or biotrophic hyphae. We then used Agrobacterium-mediated transformation to transiently express them as N-terminal fusions with fluorescent proteins in cells of Nicotiana benthamiana for imaging by confocal microscopy. Plant compartments labeled by the fusion proteins in N. benthamiana were validated by co-localization with specific organelle markers, by transient expression of the proteins in the true host plant, Arabidopsis thaliana, and by transmission electron microscopy-immunogold labeling. Among those proteins for which specific subcellular localizations could be verified, nine were imported into plant nuclei, three were imported into the matrix of peroxisomes, three decorated cortical microtubule arrays and one labeled Golgi stacks. Two peroxisome-targeted proteins harbored canonical C-terminal tripeptide signals for peroxisome import via the PTS1 (peroxisomal targeting signal 1) pathway, and we showed that these signals are essential for their peroxisome localization. Our findings provide valuable information about which host processes are potentially manipulated by this pathogen, and also reveal plant peroxisomes, microtubules, and Golgi as novel targets for fungal effectors.</p

Tree of the amino acid sequences of C-domains of strains GPE PC73, XaS3, X11-5A, BAI3, and BLS256 together with C-domains identified by Rausch et al. as starter C-domains or as dual C/E-domains (Additional file 2).

The tree was constructed using the maximum likelihood method and GTR as substitution model. Bootstrap percentages retrieved in 100 replications are shown at the main nodes. The scale bar (0.2) indicates the number of amino acid substitutions per site. C-domains belonging to the same clade as dual C/E-domains are in blue. C-domains belonging to the same clade as starter C-domains are in red. Putative starter C-domains of the loci META-A and META-C of strain GPE PC73, the contig G111 of strain XaS3 and the locus of strain BTAi similar to META-A and META-C are in green. C-domains Ax, Bx ad Cx correspond to C-domains of modules of the loci META-A, META-B and META-C of strain GPE PC73, respectively. C-domains Ox correspond to C-domains of modules of the locus META-B of strain BAI3. C-domains USxxx/x correspond to C-domains of modules of contigs of strain X11-5A. C-domains Gxxx/x correspond to C-domains of modules of contigs of strain XaS3. C-domains bradyx correspond to C-domains of the locus of Bradyrhizobium spp. strain BTAi similar to META-A and META-C (genes Bbta_6814, Bbta_6813, Bbta_6812). C-domains XOCx correspond to C-domains of the locus NRPS located in the same region as XaPPTase in strain BLS256. C-domains 0364 and 1145 correspond to C-domains of short NRPS genes XALc_0364 and XALc_1145 of strain GPE PC73, respectively. C-domain 0354XaS3 corresponds to the short NRPS gene of strain XaS3. C-domain Bbta4110 corresponds to the short NRPS gene of strain BTAi. C-domains identified by Rausch et al. [6] as starter C-domains were tagged “Starter1” to “Starter15” as follows: Starter1: Pseusyrin.NP_792633.1.m_1_leu Starter2: Pseusp.Q84BQ6.arfA_1_leu Starter3: Pseufluor.YP_259252.1.m_1_leu Starter4: Baciliche.YP_077640.1.lchAA_1_gln Starter5: Nocafarci.YP_117314.1.m_1_orn_lys_arg Starter6: Nocafarci.YP_119006.1.m_1_tyr Starter7: Nocafarci.YP_119328.1.m_1_ser Starter8: Nocafarci.YP_121279.1.m_1_ser Starter9: Strecoeli.NP_627443.1.m_1_ser Starter10: Strchrys.O68487.acmB_1_thr Starter11: Erwicarot.YP_049593.1.m_1_gln Starter12: Strprist.Q54959.snbC_1_thr Starter13: Bacisubti.NP_388230.1.srfAA_1_glu Starter14: Bacisubti.NP_389716.1.ppsA_1_glu Starter15: Baciliche.YP_090052.1.m_1_gln C-domains identified by Rausch et al. [6] as Dual C/E-domains were tagged “DualC/E1” to “DualC/E18” as follows: DualC/E1:Photlumin.NP_929905.1.m_9_thr_TO_val DualC/E2:Photlumin.NP_930489.1.m_2_val_TO_trp DualC/E3:Photlumin.NP_929905.1.m_6_bht_TO_trp DualC/E4:Bradjapon.NP_768748.1.m_3_ser_TO_phe DualC/E5:Chroviola.NP_902472.1.m_3_val_TO_ile_dual DualC/E6:Chroviola.NP_902472.1.m_1_thr_dual DualC/E7:Burkmalle.YP_106216.1.m_2_glu_TO_gly DualC/E8:Burkpseud.YP_111641.1.m_3_thr_TO_leu DualC/E9:Burkpseud.YP_111641.1.m_1_glu_gln DualC/E10:Pseusyrin.NP_792633.1.m_2_leu_TO_leu DualC/E11:Ralssolan.NP_522203.1.m_3_ser_TO_gly DualC/E12:Ralssolan.NP_522203.1.m_1_val DualC/E13:Pseufluor.YP_259253.1.m_4_leu_TO_ser DualC/E14:Pseufluor.YP_259253.1.m_2_thr_TO_ile DualC/E15:Pseusyrin.NP_792634.1.m_3_thr_TO_val DualC/E16:Pseusyrin.NP_792634.1.m_5_leu_TO_leu DualC/E17:Erwicarot.YP_049592.1.m_4_ser_TO_tyr_bht DualC/E18:Erwicarot.YP_049593.1.m_2_gln_TO_as