Host-pathogen interaction between pitaya and Neoscytalidium dimidiatum reveals the mechanisms of immune response associated with defense regulators and metabolic pathways

Abstract

Background: Understanding how plants and pathogens regulate each other's gene expression during their interactions is key to revealing the mechanisms of disease resistance and controlling the development of pathogens. Despite extensive studies on the molecular and genetic basis of plant immunity against pathogens, the influence of pitaya immunity on N. dimidiatum metabolism to restrict pathogen growth is poorly understood, and how N. dimidiatum breaks through pitaya defenses. In this study, we used the RNA-seq method to assess the expression profiles of pitaya and N. dimidiatum at 4 time periods after interactions to capture the early effects of N. dimidiatum on pitaya processes.

Results: The study defined the establishment of an effective method for analyzing transcriptome interactions between pitaya and N. dimidiatum and to obtain global expression profiles. We identified gene expression clusters in both the host pitaya and the pathogen N. dimidiatum. The analysis showed that numerous differentially expressed genes (DEGs) involved in the recognition and defense of pitaya against N. dimidiatum, as well as N. dimidiatum's evasion of recognition and inhibition of pitaya. The major functional groups identified by GO and KEGG enrichment were responsible for plant and pathogen recognition, phytohormone signaling (such as salicylic acid, abscisic acid). Furthermore, the gene expression of 13 candidate genes involved in phytopathogen recognition, phytohormone receptors, and the plant resistance gene (PG), as well as 7 effector genes of N. dimidiatum, including glycoside hydrolases, pectinase, and putative genes, were validated by qPCR. By focusing on gene expression changes during interactions between pitaya and N. dimidiatum, we were able to observe the infection of N. dimidiatum and its effects on the expression of various defense components and host immune receptors.

Conclusion: Our data show that various regulators of the immune response are modified during interactions between pitaya and N. dimidiatum. Furthermore, the activation and repression of these genes are temporally coordinated. These findings provide a framework for better understanding the pathogenicity of N. dimidiatum and its role as an opportunistic pathogen. This offers the potential for a more effective defense against N. dimidiatum.

Keywords: Host–pathogen interaction; N.dimidiatum; Pitaya canker; Transcriptomics.

Fig. 1

Flow chart of the transcriptome and overall data report for the interaction between pitaya and N. dimidiatum. A Spore suspensions were sprayed on pitaya shoots, and infected pitaya samples were collected at 5 days, 8 days, 11 days, and 15 days after spraying for cDNA library preparation and sequencing, respectively. B The circos plot shows gene expression values per kilobase of the transcript. C The heat map displays the correlation of expression between samples. The horizontal and vertical coordinates of the plot are sample numbers, the order of which is determined by the sample correlation clustering results, and the top and right sides of the plot are the corresponding clustering trees; the colors reflect the magnitude of correlation between samples. D The image shows the principal component analysis (PCA) of the pitaya

Fig. 2

The number of DEGs identified at various stages of the interaction between pitaya and N. dimidiatum. A The number of DEGs in pitaya identified at different stages of the interaction with N. dimidiatum. The overlapping circles indicate the DEGs shared among different groups. Hp0: the number of DEGs for non-infected pitaya. ND5, ND8, ND11, and ND15 represent the 5, 8, 11, and 15 days of the pitaya-N. dimidiatum interaction, respectively. B The number of DEGs in N. dimidiatum identified at different stages of the interaction with pitaya. CK0: the number of DEGs for N. dimidiatum spores. C The number of upregulated and downregulated genes in pitaya during the interaction stage between pitaya and N. dimidiatum. The blue color indicates the number of down-regulated genes; the red color indicates the number of up-regulated genes. D The number of upregulated and downregulated genes in N. dimidiatum during the interaction stage between pitaya and N. dimidiatum

Fig. 3

Pitaya infected with N. dimidiatum Go enrichment and heatmap analysis at 5 days. A GO enrichment of pitaya infested with N. dimidiatum at 5 d. B Pitaya infected with N. dimidiatum by phytopathogenic fungi interactions heatmap analysis at 5 d

Fig. 4

Identification and functional characterization of the differentially expressed genes at 8 days. A GO enrichment of pitaya infected with N. dimidiatum. B KEGG enrichment of pitaya infected with N. dimidiatum. C Heatmap analysis of plant-pathogen interaction genes when pitaya was infected with N. dimidiatum at different stages. D Expression profile and KEGG pathway analysis of ethylene and jasmonic acid-related genes involved in pathogen defense. (The KEGG pathway that the image comes from MAPK signaling pathway—plant (ko04016). Permission to use and adapt this image has been granted by Kanehisa Laboratories.)

Fig. 5

Identification and functional characterization of the DEGs at 11 days. A Go Enrichment analysis of pitaya DEGs at 11 days. B Heatmap of pectinase gene expression at different stages. C Heatmap of enrichment to xyloglucan endotransglucosylase gene expression at different stages. D Plant stomatal development-related gene expression and related pathways

Fig. 6

Heatman analysis of pitaya defense-related genes at different stages of pitaya infected by N. dimidiatum

Fig. 7

WGCNA analysis of pitaya at different interaction stages with N. dimidiatum. A Hierarchical cluster trees showing the co-expression modules identified by WGCNA. B WGCNA co-expression modules. Correlation of modules (left) and features (bottom). Red and blue represent positive and negative correlations, respectively

Fig. 8

Expression heatmap and network of protein interactions of phenylpropanoid biosynthesis-related genes. A Heatmap analysis of 44 genes related to phenylpropanoid biosynthesis. B Network protein interaction map of genes related to phenylpropanoid biosynthesis and transcription factors

Fig. 9

Principal component analysis (PCA) of N. dimidiatum

Fig. 10

Gene expression and structural analysis of N. dimidiatum effector proteins. A Heat map analysis of the 19 effector proteins before and after infestation by N. dimidiatum. B Phylogenetic tree analysis of the 19 effector proteins. C Motif analysis of 19 effector proteins. D Go enrichment analysis of N. dimidiatum DEGs

Fig. 11

qRT-PCR to validate significantly differentially expressed genes. The red bar and the blue line graph represent the qRT-PCR and RNA-seq data, respectively. Data are presented as the mean ± standard error (SE). * represents a p-value < 0.05, ** represents a p-value less than 0.01

Fig. 12

Gene pathways associated with the interaction between pitaya and N. dimidiatum. Red indicates up-regulated expression, and green indicates down-regulated expression. The solid arrows indicate the direct role, and the dashed arrows indicate the profile role