An accurate assessment of host and pathogen gene expression during infection is critical for understanding the molecular aspects of host-pathogen interactions.Often,pathogen-derived transcripts are difficult to ascert...An accurate assessment of host and pathogen gene expression during infection is critical for understanding the molecular aspects of host-pathogen interactions.Often,pathogen-derived transcripts are difficult to ascertain at early infection stages owing to the unfavourable transcript representation compared to the host genes.In this study,we compare two sequencing techniques,RNAseq and enrichment sequencing(RenSeq and PenSeq)of cDNA,to investigate gene expression patterns in the doubled monoploid potato(DM)infected with the late blight pathogen Phytophthora infestans.Our results reveal distinct advantages of cDNA RenSeq and PenSeq over traditional RNAseq in terms of target gene representation and transcriptional quantification at early infection stages.Throughout the infection time course,cDNA enrichment sequencing enables transcriptomic analyses for more targeted host and pathogen genes.For highly expressed genes that were sampled in parallel by both cDNA enrichment and RNAseq,a high level of concordance in expression profiles is observed,indicative of at least semi-quantitative gene expression representation following enrichment.展开更多
Potato is the thirdmost important food crop in theworld.Diverse pathogens threaten sustainable crop production but can be controlled,in many cases,through the deployment of disease resistance genes belonging to the fa...Potato is the thirdmost important food crop in theworld.Diverse pathogens threaten sustainable crop production but can be controlled,in many cases,through the deployment of disease resistance genes belonging to the family of nucleotide-binding,leucine-rich-repeat(NLR)genes.To identify effective disease resistance genes in established varieties,we have successfully established SMRT-AgRenSeq in tetraploid potatoes and have further enhanced the methodology by including dRenSeq in an approach that we term SMR-AgRenSeq-d.The inclusion of dRenSeq enables the filtering of candidates after the association analysis by establishing a presence/absence matrix across resistant and susceptible varieties that is translated into an F1 score.Using a SMRT-RenSeq-based sequence representation of the NLRome from the cultivar Innovator,SMRT-AgRenSeq-d analyses reliably identified the late blight resistance benchmark genes Rpi-R1,Rpi-R2-like,Rpi-R3a,and Rpi-R3b in a panel of 117 varieties with variable phenotype penetrations.All benchmark genes were identified with an F1 score of 1,which indicates absolute linkage in the panel.This method also identified nine strong candidates for Gpa5 that controls the potato cyst nematode(PCN)species Globodera pallida(pathotypes Pa2/3).Assuming that NLRs are involved in controlling many types of resistances,SMRT-AgRenSeq-d can readily be applied to diverse crops and pathogen systems.展开更多
More than 170 years after causing the potato famine in Ireland,late blight is still considered one of the most devastating crop diseases.Commercial potato breeding efforts depend on natural sources of resistance to pr...More than 170 years after causing the potato famine in Ireland,late blight is still considered one of the most devastating crop diseases.Commercial potato breeding efforts depend on natural sources of resistance to protect crops from the rapidly evolving late blight pathogen,Phytophthora infestans.We have identified and mapped a novel broad-spectrum disease resistance gene effective against P.infestans from the wild,diploid potato species Solanum bulbocastanum.Diagnostic resistance gene enrichment sequencing(dRenSeq)was used to confirm the uniqueness of the identified resistance.RenSeq and GenSeq-based mapping of the resistance,referred to as Rpi-blb4,alongside recombinant screening,positioned the locus responsible for the resistance to potato chromosome 5.The interval spans approximately 2.3 Mb and corresponds to the DM reference genome positions of 11.25 and 13.56 Mb.展开更多
Host-pathogen co-evolution shapes resistance(R)proteins and their recognition of pathogen avirulence factors.However,little attention has been paid to naturally occurring genetic diversity in R genes.In this study,12 ...Host-pathogen co-evolution shapes resistance(R)proteins and their recognition of pathogen avirulence factors.However,little attention has been paid to naturally occurring genetic diversity in R genes.In this study,12 Solanum bulbocastanum accessions from the Commonwealth Potato Collection were screened for resistance to Phytophthora infestans,identifying 11 resistant and one susceptible accession.Targeted enrichment sequencing of nucleotide-binding leucine-rich repeat(NLR)genes using RenSeq,followed by diagnostic RenSeq(dRenSeq)analysis,revealed that all accessions except 7650 contained Rpi-blb1/RB variants.Variants in accessions 7641 and 7648 were non-functional,while three novel functional variants were identified.Cloning and functional analysis of Rpi-blb1/RB variants assessed their recognition of the avirulence factor IPI-O1.Three variants were functional,conferring resistance to P.infestans.Variants in accessions 7644 and 7647 also recognized IPI-O4,confirmed in transgenic potatoes.Analysis of a non-functional variant in S.bulbocastanum accession 7648 identified amino acid Ser347 in the nucleotide-binding(NB-ARC)domain as critical for cell-death initiation following IPI-O1 recognition.Predictions from the FunFOLD2 protein-ligand interaction model suggested that Ser347 is essential for ATP binding,suggesting potential inhibition on pentameric resistosome assembly.Western blot analysis revealed that the mutation of Ser347 to Asn markedly compromises the Rpi-blb1/RB protein stability,and co-immunoprecipitation assay further confirmed that this mutation severely disrupts the self-association of CCNB,thereby preventing Rpi-blb1/RB activation.Consistently,substituting Asn347 with serine restored function,underscoring its key role in Rpi-blb1/RB activity.Cell biology experiments demonstrated that Rpi-blb1/RB relocalize to the plasma membrane in response to IPI-O1.This relocalization depends on Ser347,further supporting the idea that its mutation affects resistosome formation,impairing resistance.This study provides an in-depth functional analysis of natural Rpi-blb1/RB diversity,offering insights into NLR protein evolution and resistance mechanisms in potatoes.展开更多
基金supported by the Rural & Environment Science & Analytical Services (RESAS) Division of the Scottish Government through project JHI-B1-1the Biotechnology and Biological Sciences Research Council (BBSRC) through awards BB/ S015663/1+2 种基金BB/X009068/1Research Leaders 2025 fellowship funded by European Union’s Horizon 2020 research and innovation programme under Marie Sklodowska-Curie grant agreement no. 754380the Research/Scientific Computing teams at The James Hutton Institute and NIAB for providing computational resources and technical support for the “UK’s Crop Diversity Bioinformatics HPC” (BBSRC grant BB/ S019669/1)。
文摘An accurate assessment of host and pathogen gene expression during infection is critical for understanding the molecular aspects of host-pathogen interactions.Often,pathogen-derived transcripts are difficult to ascertain at early infection stages owing to the unfavourable transcript representation compared to the host genes.In this study,we compare two sequencing techniques,RNAseq and enrichment sequencing(RenSeq and PenSeq)of cDNA,to investigate gene expression patterns in the doubled monoploid potato(DM)infected with the late blight pathogen Phytophthora infestans.Our results reveal distinct advantages of cDNA RenSeq and PenSeq over traditional RNAseq in terms of target gene representation and transcriptional quantification at early infection stages.Throughout the infection time course,cDNA enrichment sequencing enables transcriptomic analyses for more targeted host and pathogen genes.For highly expressed genes that were sampled in parallel by both cDNA enrichment and RNAseq,a high level of concordance in expression profiles is observed,indicative of at least semi-quantitative gene expression representation following enrichment.
基金supported by the Rural&Environment Science&Analytical Services(RESAS)Division of the Scottish Government through project JHI-B1-1,the Biotechnology and Biological Sciences Research Council(BBSRC)through award BB/S015663/1 and the Royal Society through award NAF\R1\201061YW was supported through the CSC scholarship program,China.LB was supported through the East of Scotland Bioscience Doctoral Training Partnership(EASTBIO DTP)funded by the BBSRC award BB/T00875X/1.AK was supported through a Research Leaders 2025 fellowship funded by European Union’s Horizon 2020 research and innovation programme under Marie Sklodowska-Curie grant agreement no.754380.
文摘Potato is the thirdmost important food crop in theworld.Diverse pathogens threaten sustainable crop production but can be controlled,in many cases,through the deployment of disease resistance genes belonging to the family of nucleotide-binding,leucine-rich-repeat(NLR)genes.To identify effective disease resistance genes in established varieties,we have successfully established SMRT-AgRenSeq in tetraploid potatoes and have further enhanced the methodology by including dRenSeq in an approach that we term SMR-AgRenSeq-d.The inclusion of dRenSeq enables the filtering of candidates after the association analysis by establishing a presence/absence matrix across resistant and susceptible varieties that is translated into an F1 score.Using a SMRT-RenSeq-based sequence representation of the NLRome from the cultivar Innovator,SMRT-AgRenSeq-d analyses reliably identified the late blight resistance benchmark genes Rpi-R1,Rpi-R2-like,Rpi-R3a,and Rpi-R3b in a panel of 117 varieties with variable phenotype penetrations.All benchmark genes were identified with an F1 score of 1,which indicates absolute linkage in the panel.This method also identified nine strong candidates for Gpa5 that controls the potato cyst nematode(PCN)species Globodera pallida(pathotypes Pa2/3).Assuming that NLRs are involved in controlling many types of resistances,SMRT-AgRenSeq-d can readily be applied to diverse crops and pathogen systems.
基金the Rural&Environment Science&Analytical Services(RESAS)Division of the Scottish Government(JHI-B1-1)the Biotechnology and Biological Sciences Research Council(BBSRC,BB/S015663/1)+3 种基金the Royal Society(NAF\R1\201061)the National Natural Science Foundation of China(32061130211,32372558)AK was supported through a Research Leaders 2025 fellowship funded by European Union’s Horizon 2020 Research and Innovation Programme under Marie Sklodowska-Curie Grant Agreement(754380)The authors acknowledge the Research/Scientific Computing teams at The James Hutton Institute and NIAB for providing computational resources and technical support for the‘‘UK’s Crop Diversity Bioinformatics HPC”(BBSRC Grant BB/S019669/1),use of which has contributed to the results reported within this paper.
文摘More than 170 years after causing the potato famine in Ireland,late blight is still considered one of the most devastating crop diseases.Commercial potato breeding efforts depend on natural sources of resistance to protect crops from the rapidly evolving late blight pathogen,Phytophthora infestans.We have identified and mapped a novel broad-spectrum disease resistance gene effective against P.infestans from the wild,diploid potato species Solanum bulbocastanum.Diagnostic resistance gene enrichment sequencing(dRenSeq)was used to confirm the uniqueness of the identified resistance.RenSeq and GenSeq-based mapping of the resistance,referred to as Rpi-blb4,alongside recombinant screening,positioned the locus responsible for the resistance to potato chromosome 5.The interval spans approximately 2.3 Mb and corresponds to the DM reference genome positions of 11.25 and 13.56 Mb.
基金supported by the National Natural Science Foundation of China(32372558)National Natural Science Foundation of China-The Royal Society(32061130211)the Rural and Environment Science and Analytical Services Division of the Scottish Government through project JHI-B1-1,the Biotechnology and Biological Sciences Research Council(BBSRC)through award BB/SO15663/1,the Royal Society through award NAF/R1/201061.
文摘Host-pathogen co-evolution shapes resistance(R)proteins and their recognition of pathogen avirulence factors.However,little attention has been paid to naturally occurring genetic diversity in R genes.In this study,12 Solanum bulbocastanum accessions from the Commonwealth Potato Collection were screened for resistance to Phytophthora infestans,identifying 11 resistant and one susceptible accession.Targeted enrichment sequencing of nucleotide-binding leucine-rich repeat(NLR)genes using RenSeq,followed by diagnostic RenSeq(dRenSeq)analysis,revealed that all accessions except 7650 contained Rpi-blb1/RB variants.Variants in accessions 7641 and 7648 were non-functional,while three novel functional variants were identified.Cloning and functional analysis of Rpi-blb1/RB variants assessed their recognition of the avirulence factor IPI-O1.Three variants were functional,conferring resistance to P.infestans.Variants in accessions 7644 and 7647 also recognized IPI-O4,confirmed in transgenic potatoes.Analysis of a non-functional variant in S.bulbocastanum accession 7648 identified amino acid Ser347 in the nucleotide-binding(NB-ARC)domain as critical for cell-death initiation following IPI-O1 recognition.Predictions from the FunFOLD2 protein-ligand interaction model suggested that Ser347 is essential for ATP binding,suggesting potential inhibition on pentameric resistosome assembly.Western blot analysis revealed that the mutation of Ser347 to Asn markedly compromises the Rpi-blb1/RB protein stability,and co-immunoprecipitation assay further confirmed that this mutation severely disrupts the self-association of CCNB,thereby preventing Rpi-blb1/RB activation.Consistently,substituting Asn347 with serine restored function,underscoring its key role in Rpi-blb1/RB activity.Cell biology experiments demonstrated that Rpi-blb1/RB relocalize to the plasma membrane in response to IPI-O1.This relocalization depends on Ser347,further supporting the idea that its mutation affects resistosome formation,impairing resistance.This study provides an in-depth functional analysis of natural Rpi-blb1/RB diversity,offering insights into NLR protein evolution and resistance mechanisms in potatoes.
