Probing the unknowns in cytokinin-mediated immune defense in Arabidopsis with systems biology approaches

Abstract

Plant hormones involving salicylic acid (SA), jasmonic acid (JA), ethylene (Et), and auxin, gibberellins, and abscisic acid (ABA) are known to regulate host immune responses. However, plant hormone cytokinin has the potential to modulate defense signaling including SA and JA. It promotes plant pathogen and herbivore resistance; underlying mechanisms are still unknown. Using systems biology approaches, we unravel hub points of immune interaction mediated by cytokinin signaling in Arabidopsis. High-confidence Arabidopsis protein-protein interactions (PPI) are coupled to changes in cytokinin-mediated gene expression. Nodes of the cellular interactome that are enriched in immune functions also reconstitute sub-networks. Topological analyses and their specific immunological relevance lead to the identification of functional hubs in cellular interactome. We discuss our identified immune hubs in light of an emerging model of cytokinin-mediated immune defense against pathogen infection in plants.

Keywords: cytokinin; gene expression; interaction networks; plant hormones; systems biology.

Figure 1

A model of cytokinin responses in plant immunity and systems biology tools for the identification of cytokinin-linked immune regulators. (A) Model and hypothesis. Cytokinin promotes plant immunity against infection with some of the biotrophic (Hpa Noco2), necrotrophic (Alternaria), as well as hemi-biotrophic pathogens (boxes, arrows). The presence of cytokinin is perceived by membrane-spanning receptors AHKs (plasma membrane or ER membrane may house AHKs) and transmit the phosphorely signal to type-B ARRs via AHPs. Type-B ARR, ARR2 interacts with TGA transcription factors and promotes SA signaling for resistance against infection with Pst DC3000 (hemi-biotroph). type-A ARRs, which are negative regulators of cytokinin signaling, are transcriptionally activated by type-B ARRs. Deletion of type-A ARRs enhances cytokinin signaling and derepresses SA responses in promoting Arabidopsis immunity against infection with Hpa Noco2 (biotroph). Enhanced cytokinin levels and signaling also promotes Arabidopsis resistance to infection with Alternaria (necrotroph). The potential link between cytokinin and JA pathway is still unknown. The signal of cytokinin is fortified through enhanced endogenous production by overexpressing IPT enzyme gene or by external application of cytokinin. Also, deletion of type-A ARRs relives its inhibition to cytokinin signaling. The impact of exogenous application of cytokinin and deletion of type-A ARRs on plant immune defense is further analyzed by adopting systems biology approaches. (B) Methodology to address hypothesis. Transcriptome data on the exogenous application of trans-Zeatin and type-A ARRs sextuplet mutants are collected from GEO and studies of Argueso et al., respectively (green files). High-confidence (edge score < 0.9) PPI from the STRING database are mapped and visualized with Cytoscape. DEGs from transcriptomes and their corresponding proteins in interactome are identified as intersecting coverage between the two levels of omics data (transcriptome × interactome). Intersected nodes (green dots) are subjected to functional enrichment analysis by using Cytoscape plugin BiNGO. Proteins of the network relevant to plant immune defense processes (red dots) are considered as immune nodes of the network. Immune networks are composed of immune-enriched nodes (red dots) as well as their interactors (black dots) from the basal interactome. Taking the degree of connectivity and neighborhood (network analysis) as well as biological relevance to immune defense as parameters, we then looked for regulatory hubs mediated by cytokinin signaling in Arabidopsis.

Figure 2

Systems biology analysis for the assessment of the impact of exogenous cytokinin application on plant immune defense in Arabidopsis. (A) Transcriptome × interactome mapping and visualization. Proteins are indicated by little squares. The color for non-intersecting nodes between transcriptome and interactome is cyan, for intersecting up-regulated nodes between transcriptome and interactome is violet, and for intersecting down-regulated nodes between transcriptome and interactome is dark blue; interactions are drawn as gray lines. Transcriptome changes due to the exogenous application of cytokinin in comparison to mock application in Arabidopsis are collected from GEO (GSE6832.I). GEO2r-based normalization of selected samples resulted in 5489 DEGs (logFC < ±1). High-confidence interactome (<0.9 as combined edge score) of Arabidopsis was acquired from STRING (3056 protein nodes, 18233 interactions) and visualized with Cytoscape, version 2.8.3 (www.cytoscape.org). 575 nodes are shared between interactome and transcriptome (327 up regulated: violet, 248 down regulated: dark blue). (B) Biological function analysis and reconstruction of the immune network. 10 functional hubs were identified and are shown as bold red dots with numbers from 1 to 10 on the rim of the immune network. Overall, there are 130 nodes with immune enrichment (red little circles on the rim) and 525 immune interactors (light blue circles). The latter are overlapping and covering the rim. 1039 interactions between all proteins are given in gray and cross the interior of the figure. The 575 shared nodes were analyzed with Cytoscape plugin tool BiNGO to detect over-represented biological functions in the interactome adapted to the cytokinin responses. We identified 363 biological functions for 501 genes; 74 genes had no BiNGO-based annotation (see supplementary material). We only focused on 35 immune and defense-related terms (see Supplementary Table 1). 130 protein nodes of the intersecting interactome participating in these 35 immune functions form a base for the reconstruction of the immune network. (C) Network analysis and identification of functional hubs. Topological analysis of immune network (B): Network properties such as number of degree and average number of neighbors were next computed applying the Cytoscape plugin NetworkAnalyzer. The double logarithmic plot shows (black dots) for each degree (number of connection to other nodes, x-axis), the number of nodes found (y-axis). The average degree is shown as red vertical line. Well-connected proteins (“hubs”) with degree equal to 7 or higher are framed in bold. Based on network connectivity and topology, expression P-values, and immunological relevance, 10 functional hubs are identified and shown as bold red dots with numbers from 1 to 10 in the immune network (B). They are tabulated with quantitative details in (D).

Figure 3

Systems biology analysis to determine the effect of derepression of cytokinin signaling on plant immunity. Type-A ARRs attenuate cytokinin signaling. Hence, plant mutants deficient in type-A ARRs enhance cytokinin responses including pathogen defense (color code as in Fig. 2). (A) Transcriptome × interactome mapping and visualization. Proteins are indicated by little squares. The color for non-intersecting nodes between transcriptome and interactome is cyan, for intersecting up-regulated nodes between transcriptome and interactome is violet, and for intersecting down-regulated nodes between transcriptome and interactome is dark blue; interactions are drawn as gray lines. Transcriptome data of sextuplet type-A ARRs mutant (arr3,4,5,6,8,9) Arabidopsis plants vs. wild-type Col-0 and Hpa Noco2 treated vs. mock treated are adopted from the study of Argueso et al. 1577 genes are differentially regulated. Interactome of Arabidopsis (3056 protein nodes, 18233 interactions), acquired from STRING, is visualized with Cytoscape. 227 nodes are shared between transcriptome and interactome data sets (145 up regulated: violet, 82 down regulated: dark blue). (B) Biological function analysis and reconstruction of immune network. Functional hubs are shown as bold red dots on the rim, immune enrichment as red little circles on the rim, interacting proteins as overlapping light blue circles on the rim, and protein interactions as gray lines traversing the circle to find the protein partner. Overrepresented biological functions in the interactome adaptation to the pathogen are detected by the Cytoscape plugin BiNGO tool. 414 biological functions for 214 genes are identified, 13 genes had no BiNGO-based annotation (see supplementary material), and 43 immune process-related protein nodes participate in 15 immune functions (red ones, see supplementary Table 1). The reconstructed immune network of these proteins and their interactors (light blue, maintained from step a) consists of 168 nodes and 261 edges (gray). (C) The double logarithmic plot shows (black dots) for each degree (number of connection to other nodes, x-axis), the number of nodes found (y-axis). The average degree is shown as red vertical line. Well-connected proteins (“hubs”) with degree equal to 7 or higher is framed in bold. Based on network connectivity and topology, expression P-values, and immunological relevance, 10 functional hubs are identified and shown as bold red dots with numbers from 1 to 10 in the immune network (B). They are tabulated with quantitative details in (D).

Figure 4

An integrative view on modulations in cytokinin pathway at two different levels and their impact on immune regulatory hubs in Arabidopsis. (A) Localization of hubs due to the deletion of type-A ARRs (big dark green squares) compared to hubs due to the exogenous application of cytokinins (big yellow squares). The hub nodes are projected on the background of all nodes from the high-confidence interactome (proteins: small light green squares; interactions: gray lines). (B) Compact interactome and short, direct interaction paths (blue lines; strength: confidence level) between hub protein nodes (colored, drop-like circles) are observed after the deletion of type-A ARRs. This was drawn according to information from STRING database. (C) Complex, non-compact connectivity paths (blue lines; strength: confidence level) among hub proteins (colored, drop-like circles) affected after the exogenous application of cytokinins. This was drawn according to information from STRING database.