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. 2021 May 2;10(5):913.
doi: 10.3390/plants10050913.

Identification of Susceptibility Genes in Castanea sativa and Their Transcription Dynamics following Pathogen Infection

Vera Pavese  1 , Andrea Moglia  1 , Paolo Gonthier  1 , Daniela Torello Marinoni  1 , Emile Cavalet-Giorsa  1 , Roberto Botta  1
Affiliations

Identification of Susceptibility Genes in Castanea sativa and Their Transcription Dynamics following Pathogen Infection

Vera Pavese et al. Plants (Basel). .

Abstract

Castanea sativa is one of the main multipurpose tree species valued for its timber and nuts. This species is susceptible to two major diseases, ink disease and chestnut blight, caused by Phytophthora spp. and Cryphonectria parasitica, respectively. The loss-of-function mutations of genes required for the onset of pathogenesis, referred to as plant susceptibility (S) genes, are one mechanism of plant resistance against pathogens. On the basis of sequence homology, functional domain identification, and phylogenetic analyses, we report for the first time on the identification of S-genes (mlo1, dmr6, dnd1, and pmr4) in the Castanea genus. The expression dynamics of S-genes were assessed in C. sativa and C. crenata plants inoculated with P. cinnamomi and C. parasitica. Our results highlighted the upregulation of pmr4 and dmr6 in response to pathogen infection. Pmr4 was strongly expressed at early infection phases of both pathogens in C. sativa, whereas in C. crenata, no significant upregulation was observed. The infection of P. cinnamomi led to a higher increase in the transcript level of dmr6 in C. sativa compared to C. crenata-infected samples. For a better understanding of plant responses, the transcript levels of defense genes gluB and chi3 were also analyzed.

Keywords: Cryphonectria parasitica; Phytophthora cinnamomi; chestnut; susceptibility genes.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Chestnut S-genes and their protein structures. The graphical representations of gene exon/intron structures were generated using the http://wormweb.org/exonintron tool (accessed on 31 March 2021) and are shown in the left panel. The exons are indicated with black boxes, whereas introns are shown with lines. In the right panel, the protein structural domains are displayed.
Figure 2
Figure 2
Phylogenetic analysis of the S-genes. The 4 phylogenetic trees of mlo1 (A), dnd1 (B), pmr4 (C), and dmr6 (D) were constructed using MEGAX software by aligning chestnut S-gene coding sequences with NCBI S-gene ortholog coding sequences (available in S3 File). The colors indicate the main clades detected, and the arrows underline the location of C. mollissima. To visualize details, all the phylogenetic trees are available in S4 File.
Figure 3
Figure 3
The DMR6 3D protein model created using Modeller software and visualized using Ccp4mg software. The C. mollissima (Cm) DMR6 (blue) protein and A. thaliana (At) DMR6 (yellow) protein are shown.
Figure 4
Figure 4
qRT-PCR pathogen DNA quantification after P. cinnamomi inoculation. Data were quantified using the 2−ΔΔCt method based on the Ct values of pathogen genes (ypt and mf1) and actin-7 as a housekeeping gene. Data are the means of three biological replicates ± SE. C. sativa data are normalized with C. sativa 0 hpi control; C. crenata data are normalized with C. crenata 0 hpi control. Different letters associated with the set of means indicate a significant difference based on Tukey’s HSD test (p ≤ 0.05).
Figure 5
Figure 5
qRT-PCR-based transcription profiling after P. cinnamomi inoculation. (A) The S-gene transcription profiles in C. sativa (blue) and C. crenata (green) chestnut species. (B) The expression analysis of genes coding for several pathogenesis-related (PR) proteins in C. sativa (blue) and C. crenata (green) species. In all analyses, Cm7-actin was used as a housekeeping gene. Data are the means of three biological replicates ± SE. C. sativa data are normalized with C. sativa 0 hpi control; C. crenata data are normalized with C. crenata 0 hpi control. Different letters associated with the set of means indicate a significant difference based on Tukey’s HSD test (p ≤ 0.05).
Figure 6
Figure 6
qRT-PCR pathogen DNA quantification after C. parasitica inoculation. Data were quantified using the 2−ΔΔCt method based on the Ct values of fungal genes (ypt and mf1) with actin-7 as a housekeeping gene. Data are the means of three biological replicates ± SE. C. sativa data are normalized with the C. sativa 0 hpi control; C. crenata data are normalized with C. crenata 0 hpi control. Different letters associated with the set of means indicate a significant difference based on Tukey’s HSD test (p ≤ 0.05).
Figure 7
Figure 7
qRT-PCR-based transcription profiling after C. parasitica inoculation. (A) The S-gene transcription profile in C. sativa (blue) and C. crenata (green) chestnut species. (B) The expression analysis of genes coding for several pathogenesis-related (PR) proteins of C. sativa (blue) and C. crenata (green) species. In all the analyses, Cm7-actin was used as the housekeeping gene. The data are the means of three biological replicates ± SE. C. sativa data are normalized with C. sativa 0 hpi control; C. crenata data are normalized with C. crenata 0 hpi control. Different letters associated with the set of means indicate a significant difference based on Tukey’s HSD test (p ≤ 0.05).
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