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6. Nitrate transporter protein NPF5.12 and major latex-like protein MLP6 are important defense factors against Verticillium longisporum.

Author

Dölfors, Fredrik, Ilbäck, Jonas, Bejai, Sarosh, Fogelqvist, Johan, and Dixelius, Christina

Subjects
CARRIER proteins, RAPESEED, VERTICILLIUM, NITRATES, NITRATE reductase, FUNGAL growth
Abstract

Plant defense responses to the soil-borne fungus Verticillium longisporum causing stem stripe disease on oilseed rape (Brassica napus) are poorly understood. In this study, a population of recombinant inbred lines (RILs) using the Arabidopsis accessions Sei-0 and Can-0 was established. Composite interval mapping, transcriptome data, and T-DNA mutant screening identified the NITRATE/PEPTIDE TRANSPORTER FAMILY 5.12 (AtNPF5.12) gene as being associated with disease susceptibility in Can-0. Co-immunoprecipitation revealed interaction between AtNPF5.12 and the MAJOR LATEX PROTEIN family member AtMLP6, and fluorescence microscopy confirmed this interaction in the plasma membrane and endoplasmic reticulum. CRISPR/Cas9 technology was applied to mutate the NPF5.12 and MLP6 genes in B. napus. Elevated fungal growth in the npf5.12 mlp6 double mutant of both oilseed rape and Arabidopsis demonstrated the importance of these genes in defense against V. longisporum. Colonization of this fungus depends also on available nitrates in the host root. Accordingly, the negative effect of nitrate depletion on fungal growth was less pronounced in Atnpf5.12 plants with impaired nitrate transport. In addition, suberin staining revealed involvement of the NPF5.12 and MLP6 genes in suberin barrier formation. Together, these results demonstrate a dependency on multiple plant factors that leads to successful V. longisporum root infection. [ABSTRACT FROM AUTHOR]

Published
2024
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25. Rhizoctonia solani Infection Assay of Young Sugar Beet and Arabidopsis plantlets

Author

Dölfors, Fredrik, Holmquist, Louise, Tzelepis, Georgios, and Dixelius, Christina

Subjects
Strategy and Management, Mechanical Engineering, Methods Article, Metals and Alloys, Industrial and Manufacturing Engineering
Abstract

Rhizoctonia solani is a soil-borne fungus, which rarely produces any spores in culture. Hence, all inoculation procedures are based on mycelia, often as a coat on cereal kernels, placed in close vicinity to the plant to be infected. In this protocol, an inoculation method is described where the fungus is first allowed to infest a perlite-maize flour substrate for 10 days, followed by thorough soil mixing to generate uniform fungal distribution. Pre-grown seedlings are then replanted in the infested soil. Plant materials can be harvested, five (sugar beet) and ten days (Arabidopsis) post infection, followed by a rapid cleaning step ahead of any nucleic acid preparation. Commercial DNA or RNA extraction kits can be used or, if higher DNA yield is required, a CTAB extraction method. Our purpose was to develop a reliable and reproducible protocol to determine the infection levels in planta upon infection with R. solani. This protocol is less laborious compared to previous ones, improves the consistency of plant infection, reproducibility between experiments, and suits both a root crop and Arabidopsis. Graphic abstract: [Image: see text] Plant infectionInoculation of R. solaniPreparation of inoculumPCR analysis Overview of the R. solani infection procedure.

Published
2022
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33. Phytophthora infestans Ago1-associated miRNA promotes potato late blight disease

Author

Hu, Xinyi, Persson Hodén, Kristian, Liao, Zhen, Åsman, Anna, and Dixelius, Christina

Subjects
fungi, food and beverages, Agricultural Science
Abstract

Phytophthora spp. cause serious damage to plants by exploiting a large number of effector proteins and small RNAs (sRNAs). Several reports have described modulation of host RNA biogenesis and defence gene expression. Here, we analysed Phytophthora infestans Argonaute (Ago) 1 associated small RNAs during potato leaf infection. Small RNAs were co-immunoprecipitated, deep sequenced and analysed against the P. infestans and potato genomes, followed by transcript analyses and transgenic assays on a predicted target. Extensive targeting of potato and pathogen-derived sRNAs to a range of mRNAs was observed, including 638 sequences coding for resistance (R) proteins in the host genome. The single miRNA encoded by P. infestans (miR8788) was found to target a potato alpha/beta hydrolase-type encoding gene (StABH1), a protein localized to the plasma membrane. Analyses of stable transgenic potato lines harbouring overexpressed StABH1 or artificial miRNA gene constructs demonstrated the importance of StABH1 during infection by P. infestans. miR8788 knock-down strains showed reduced growth on potato, and elevated StABH1 expression levels were observed when plants were inoculated with the two knock-down strains compared to the wild-type strain 88069. The findings of our study suggest that sRNA encoded by P. infestans can affect potato mRNA, thereby expanding our knowledge of the multifaceted strategies this species uses to facilitate infection.

Published
2021

47. Phytophthora infestans Ago1‐associated miRNA promotes potato late blight disease.

Author

Hu, Xinyi, Persson Hodén, Kristian, Liao, Zhen, Åsman, Anna, and Dixelius, Christina

Subjects
LATE blight of potato, PHYTOPHTHORA infestans, MICRORNA, NON-coding RNA, CELL membranes, POTATOES
Abstract

Summary: Phytophthora spp. cause serious damage to plants by exploiting a large number of effector proteins and small RNAs (sRNAs). Several reports have described modulation of host RNA biogenesis and defence gene expression. Here, we analysed Phytophthora infestans Argonaute (Ago) 1 associated small RNAs during potato leaf infection.Small RNAs were co‐immunoprecipitated, deep sequenced and analysed against the P. infestans and potato genomes, followed by transcript analyses and transgenic assays on a predicted target.Extensive targeting of potato and pathogen‐derived sRNAs to a range of mRNAs was observed, including 638 sequences coding for resistance (R) proteins in the host genome. The single miRNA encoded by P. infestans (miR8788) was found to target a potato alpha/beta hydrolase‐type encoding gene (StABH1), a protein localized to the plasma membrane. Analyses of stable transgenic potato lines harbouring overexpressed StABH1 or artificial miRNA gene constructs demonstrated the importance of StABH1 during infection by P. infestans. miR8788 knock‐down strains showed reduced growth on potato, and elevated StABH1 expression levels were observed when plants were inoculated with the two knock‐down strains compared to the wild‐type strain 88069.The findings of our study suggest that sRNA encoded by P. infestans can affect potato mRNA, thereby expanding our knowledge of the multifaceted strategies this species uses to facilitate infection. [ABSTRACT FROM AUTHOR]

Published
2022
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