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PreprintCited by 0Nematode management and characterization studiesPlant Disease Resistance and GeneticsPlant Parasitism and Resistance

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Subcellular localization and differential expression account for the function of the nematode resistance gene Hs4

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Schildberg, Annika, Dorn, Kevin, Jung, Christian·2025·Scientific Reports
DOI 10.21203/rs.3.rs-6653794/v1PMC PMC12953702

Preprint — published as 10.1038/s41598-026-40666-5 · Paper page

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Abstract

Plant-parasitic nematodes are economically important threats to global crop production. The beet cyst nematode ( Heterodera schachtii ) is a crucial pest in sugar beet ( Beta vulgaris ). While all species of the genus Beta are highly susceptible, the three species of the beet wild relative genus Patellifolia are entirely resistant. Recently, we cloned the Hs4 gene from P. procumbens , which confers complete resistance. In this study, we aimed to determine whether putative Hs4 orthologs exist in Beta and Patellifolia species. The Hs4 gene consisted of 4999 bp, with six exons and five introns. Patellifolia species contain highly similar Hs4 homologs. Single nucleotide polymorphisms and insertions/deletions between the accessions and species could be detected. We found an exonic integration of three bases, resulting in the addition of one amino acid. Interestingly, this variant was present in single accessions of all three Patellifolia species. Beta vulgaris contains an Hs4 homolog ( BvHs4 ) with 60% protein identity to Hs4. BvHs4 homologs were present in all Beta species analyzed. Further, we examined the expression patterns of Hs4 and BvHs4 homologs. While Hs4 homologs from Patellifolia species are strongly expressed in roots, BvHs4 homologs are expressed mainly in leaves. When the spatio-temporal expression of Hs4 was examined, no response to nematode inoculation was observed. These results are highly relevant for searching for functional Hs4 alleles and breeding nematode-resistant varieties.

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Fig. 1 Structure of Hs4 (2022 bp) and its Beta homolog BvHs4 (4342 bp) and the intron-less open reading frame (ORF) derived from them. Exons are shown in green (Hs4) and orange (BvHs4). The primer positions to amplify a region of both genes simultaneously (F7/R7) or the whole genes (Hs4: F5/R6; BvHs4: F1/R0) are depicted by arrowheads. The figure was generated using the Illustrator for Biological Sequences software49.
Fig. 1 Structure of Hs4 (2022 bp) and its Beta homolog BvHs4 (4342 bp) and the intron-less open reading frame (ORF) derived from them. Exons are shown in green (Hs4) and orange (BvHs4). The primer positions to amplify a region of both genes simultaneously (F7/R7) or the whole genes (Hs4: F5/R6; BvHs4: F1/R0) are depicted by arrowheads. The figure was generated using the Illustrator for Biological Sequences software49.CC BY · PubMed Central
Fig. 2 PCR amplicons of Hs4- and its BvHs4-homologous genes separated by agarose gel electrophoresis. Using the same set of primers, a single 1217 bp fragment was amplified in all Patellifolia accessions (A), while a 1499 bp fragment was generated in most Beta accessions (B). The resistant translocation lines (TR363 and TR520) and the resistant cultivar Nemata display both Hs4 and BvHs4 fragments. M: 1 kb ladder; N: negative control.
Fig. 2 PCR amplicons of Hs4- and its BvHs4-homologous genes separated by agarose gel electrophoresis. Using the same set of primers, a single 1217 bp fragment was amplified in all Patellifolia accessions (A), while a 1499 bp fragment was generated in most Beta accessions (B). The resistant translocation lines (TR363 and TR520) and the resistant cultivar Nemata display both Hs4 and BvHs4 fragments. M: 1 kb ladder; N: negative control.CC BY · PubMed Central
Fig. 3 A phylogenetic tree was created with polypeptide sequences from different Patellifolia and Beta accessions. The tree was constructed using the maximum likelihood method with MEGA1138. Bootstrapping was conducted with 500 replicates. Quinoa (C. quinoa), spinach (S. oleracea), and mung bean (V. radiata) were used as outgroups.
Fig. 3 A phylogenetic tree was created with polypeptide sequences from different Patellifolia and Beta accessions. The tree was constructed using the maximum likelihood method with MEGA1138. Bootstrapping was conducted with 500 replicates. Quinoa (C. quinoa), spinach (S. oleracea), and mung bean (V. radiata) were used as outgroups.CC BY · PubMed Central
Fig. 4 Expression levels of Hs4 and its homologs in leaves (L) and roots (R) of resistant Patellifolia (A) and susceptible Beta (B) accessions as determined by RT-qPCR (primer: AK_F7/AK_R5 for Hs4 and AS_BvHs4_F1/AS_BvHs4_R7 for BvHs4; reference gene: GAPDH). (A) Hs4 expression in fifteen Patellifolia accessions and three resistant translocation lines. In all samples, the expression was higher in the roots. (B) Expression of BvHs4 orthologs across five different Beta species. Due to a lack of biological replicates, the expression data of two different B. macrorhiza accessions were pooled in this analysis. Bars represent the standard error of the mean (SEM).
Fig. 4 Expression levels of Hs4 and its homologs in leaves (L) and roots (R) of resistant Patellifolia (A) and susceptible Beta (B) accessions as determined by RT-qPCR (primer: AK_F7/AK_R5 for Hs4 and AS_BvHs4_F1/AS_BvHs4_R7 for BvHs4; reference gene: GAPDH). (A) Hs4 expression in fifteen Patellifolia accessions and three resistant translocation lines. In all samples, the expression was higher in the roots. (B) Expression of BvHs4 orthologs across five different Beta species. Due to a lack of biological replicates, the expression data of two different B. macrorhiza accessions were pooled in this analysis. Bars represent the standard error of the mean (SEM).CC BY · PubMed Central
Fig. 5 Expression levels of Hs4 in P. procumbens (primer: AK_F7/AK_R5) and BvHs4 in B. vulgaris ssp. vulgaris (primer: AS_BvHs4_F1/AS_BvHs4_R7) determined by RT-qPCR. GAPDH was used as a reference. i: inoculated; ni: non-inoculated; R: root; H: hypocotyl; C: cotyledon; L: leaf. A Kruskal-Wallis test (p < 0.05) was performed for the P. procumbens samples, and significant differences between groups were calculated using the Hochberg-corrected Dunn post-hoc test (p < 0.05). Statistical significances between B. vulgaris ssp. vulgaris samples were calculated using a one-way ANOVA (p < 0.05), followed by the Tukey HSD Test. Standard deviations are given as SEM. The letters above the bars indicate statistically significant differences between group means; groups sharing the same letter are not significantly different.
Fig. 5 Expression levels of Hs4 in P. procumbens (primer: AK_F7/AK_R5) and BvHs4 in B. vulgaris ssp. vulgaris (primer: AS_BvHs4_F1/AS_BvHs4_R7) determined by RT-qPCR. GAPDH was used as a reference. i: inoculated; ni: non-inoculated; R: root; H: hypocotyl; C: cotyledon; L: leaf. A Kruskal-Wallis test (p < 0.05) was performed for the P. procumbens samples, and significant differences between groups were calculated using the Hochberg-corrected Dunn post-hoc test (p < 0.05). Statistical significances between B. vulgaris ssp. vulgaris samples were calculated using a one-way ANOVA (p < 0.05), followed by the Tukey HSD Test. Standard deviations are given as SEM. The letters above the bars indicate statistically significant differences between group means; groups sharing the same letter are not significantly different.CC BY · PubMed Central

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Metadata

Authors
Schildberg, A., Dorn, K., & Jung, C.
Affiliations
  • Christian-Albrechts-Universität zu Kiel (DE)
  • Agricultural Research Service (US)
Corresponding author
Jung, Christian
ORCID
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2025-06-18
Volume
16
Issue
1
ISSN
2045-2322 (ISSN-L)
DOI
10.21203/rs.3.rs-6653794/v1
PMC
PMC12953702
OpenAlex
W7132837844
Cited by
0
References
43
FWCI
0.00
Type
journal article
Language
en
Open access
Licence
Locations
  • Scientific Reports (publishedVersion, cc-by)
  • Research Square (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Deutsche Forschungsgemeinschaft
  • Christian-Albrechts-Universität zu Kiel
  • Exzellenzclusters Entzündungsforschung
Topics
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Nematode management and characterization studies
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant Disease Resistance and Genetics
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant Parasitism and Resistance
Keywords
Heterodera schachtii, Beta vulgaris, nematode resistance, subcellular localization, differential gene expression, single nucleotide polymorphisms, insertion-deletion variants, orthologs, sugar beet, root expression, spatiotemporal expression, functional alleles, plant breeding
MeSH terms
Amino Acid Sequence, Animals, Phylogeny, Plant Diseases, Plant Proteins, Tylenchoidea, Gene Expression Regulation, Plant, Polymorphism, Single Nucleotide, Beta vulgaris, Disease Resistance

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Schildberg, A., Dorn, K., & Jung, C. (2025). Subcellular localization and differential expression account for the function of the nematode resistance gene Hs4. Scientific Reports. https://doi.org/10.21203/rs.3.rs-6653794/v1

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