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Role of Exopolygalacturonase-Related Genes in Potato-Verticillium dahliae Interaction.

ABSTRACT: Verticillium dahliae is a hemibiotrophic pathogen responsible for great losses in dicot crop production. An ExoPG gene (VDAG_03463,) identified using subtractive hybridization/cDNA-AFLP, showed higher expression levels in highly aggressive than in weakly aggressive V. dahliae isolates. We used a vector-free split-marker recombination method with PEG-mediated protoplast to delete the ExoPG gene in V. dahliae. This is the first instance of using this method for V. dahliae transformation. Only two PCR steps and one transformation step were required, markedly reducing the necessary time for gene deletion. Six mutants were identified. ExoPG expressed more in the highly aggressive than in the weakly aggressive isolate in response to potato leaf and stem extracts. Its expression increased in both isolates during infection, with higher levels in the highly aggressive isolate at early infection stages. The disruption of ExoPG did not influence virulence, nor did it affect total exopolygalacturonase activity in V. dahliae. Full genome analysis showed 8 more genes related to polygalacturonase/pectinase activity in V. dahliae. Transcripts of PGA increased in the △exopg mutant in response to potato leaf extracts, compared to the wild type. The expression pattern of those eight genes showed similar trends in the △exopg mutant and in the weakly aggressive isolate in response to potato extracts, but without the increase of PGA in the weakly aggressive isolate to leaf extracts. This indicated that the △exopg mutant of V. dahliae compensated by the suppression of ExoPG by activating other related gene.

SUBMITTER: Zhu X

PROVIDER: S-EPMC8224799 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Intercropping could alleviate soil-borne diseases, however, few studies focused on the immunity of the host plant induced by the interspecific interactions. To test whether or not intercropping could enhance the disease resistance of host plant, we investigated the effect of companion cropping with potato onion on tomato Verticillium wilt caused by Verticillium dahliae (V. dahliae). To investigate the mechanisms, the root exudates were collected from tomato and potato onion which were grown together or separately, and were used to examine the antifungal activities against V. dahliae in vitro, respectively. Furthermore, RNA-seq was used to examine the expression pattern of genes related to disease resistance in tomato companied with potato onion compared to that in tomato grown alone, under the condition of infection with V. dahliae. The results showed that companion cropping with potato onion could alleviate the incidence and severity of tomato Verticillium wilt. The further studies revealed that the root exudates from tomato companied with potato onion significantly inhibited the mycelia growth and spore germination of V. dahliae. However, there were no significant effects on these two measurements for the root exudates from potato onion grown alone or from potato onion grown with tomato. RNA-seq data analysis showed the disease defense genes associated with pathogenesis-related proteins, biosynthesis of lignin, hormone metabolism and signal transduction were expressed much higher in the tomato companied with potato onion than those in the tomato grown alone, which indicated that these defense genes play important roles in tomato against V. dahliae infection, and meant that the disease resistance of tomato against V. dahliae was enhanced in the companion copping with potato onion. We proposed that companion cropping with potato onion could enhance the disease resistance of tomato against V. dahliae by regulating the expression of genes related to disease resistance response. This may be a potential mechanism for the management of soil-borne plant diseases in the intercropping system.

Project description:Purpose: Verticillium dahliae is a fungal plant pathogen. The goals of the study are to examine gene networks involved in controlling resistance to V. dahliae in potato using expression quantitative trait loci (eQTL) mapping. Methods: 91 clones from a diploid potato mapping population 15143 and the parental lines 07506-01 and 12120-03 from the Agriculture and Agri-Food Canada in Fredericton, New Brunswick, Canada were propagated in a V. dahliae-infested field in triplicate plots of five plants in a randomized complete block design. Tetraploid varieties Shepody, Hindenburg, Kennebec, Russet Burbank, Atlantic and Green Mountain were planted similarly and included in the analysis. Green Mountain was also planted in additional plots and used a guard row clone. Leaf samples were taken at 70 days after planting. Two µg of total RNA per sample was used to construct DeepSAGE tag libraries using a modification to facilitate direct sequencing of the amplicons by Illumina sequencing. The samples from 91 clones were bar-coded with a different identification key. Samples were diluted to a final concentration of 10 nM and were pooled. Each of the three biological replicates was done in a separate pool. Final pool concentrations were estimated using Quant-iT-PicoGreen prior to template DNA hybridization and sequencing on an Illumina Genome Analyzer II according to the manufacturer's instructions. Results: Tag count data were transformed to the relative unit of counts per million. Tags included in the analysis had to be present in at least one biological replicate. Clonal variation between samples was detected (p-value ≤ 0.05) using the exact test for a negative binomial distribution . Tags that did not have significant clonal variation were removed, leaving 6248 tags. The LS mean of tag count was calculated for each tag for each clone. The 21-nt tag sequences were aligned against PGSC S. tuberosum group Phureja DM1-3 transcripts (v3.4) using blastn. Hits with E-value ≤ 0.5 were filtered in. Where there were multiple blast hits meeting E-value criteria, only the one with the longest alignment was included. 4198 tags were assigned single-hit annotations in this manner and were used for eQTL analysis. Conclusions: The results indicated that for most genes the eQTL controlling expression was mapped near the gene (cis-eQTL). There were also many trans-eQTLs, where were eQTLs for a gene that were found on another chromosome particularly on chromosome 5.

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Role of Exopolygalacturonase-Related Genes in Potato-Verticillium dahliae Interaction.

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