In this study we hypothesized that different sets of genes are expressed in response to A. cochlioides invasion in susceptible and partially resistant sugar beet genotypes. Thus, we investigated changes in the transcript profiles in sugar beet breeding lines known to display different phenotypes after infection caused with the pathogen. Our aim was to identify defense-related genes that enable the plant to cope with pathogen attack during the initial stages of the infection. The major source of variation in our dataset was host genotype, with samples clustering by genotype and not by treatment. Interestingly, a recent transcriptomics investigation of the resistance responses of pea to infection by Aphanomyces euteiches [27] found a similar variation related to genotype, indicating that the gene expression governing resistance mechanisms against pathogen invasion strongly depends on the specific genotype.
General differential expressed genes correlated to stress responses in sugar beet
In order to understand the generic responses induced by A. cochlioides infection, the function of the up-regulated genes shared between the four sugar beet breeding lines analyzed in this study was investigated. Overall, all genotypes shared some differential expressed genes, corresponding to stress-related genes. These included chalcone synthases (CHS), auxin-binding proteins, glutathione S-transferases (GSTs) and germin-like proteins (GLP).
Chalcone synthase (CHS) is a key enzyme of the flavonoid/isoflavonoid biosynthesis pathway and is known to be commonly expressed under several type of stresses including bacterial and fungal infections [28]. The expression of this gene results in the production of flavonoids and isoflavonoids with antimicrobial activity, known as phytoalexins. The accumulation of these antimicrobic metabolites in response to pathogen attacks is well established and has been described in several plant species, including sugar beet [28][29]. KEGG analysis showed that genes involved in secondary metabolite pathways, and in particular in flavonoid and phenylpropanoid biosynthesis, underwent significant changes in both partially resistant and susceptible lines, indicating that they play a central role during the host-pathogen interaction.
The phytohormone auxin plays a central role not only in plant growth and development but also in plant immune signaling [30]. In A. thaliana, the auxin response pathway has been reported to be involved in resistance against oomycetes [31]. Microarray analysis on the model plant Medicago truncatula, performed to study the transcriptomic responses of pea (Pisum sativum L.) to the oomycetes Phytophthora pisi and A. euteiches, demonstrated that CHS and auxin pathways are specifically induced in response to A. euteiches infection [32]. The up-regulation of an auxin-responsive gene was also identified in a pea susceptible genotype in response to A. euteiches infection [27]. The large number of up-regulated genes corresponding to chalcone synthases and auxin-binding proteins in all sugar beet lines tested here might indicate that these pathways are also induced in sugar beet in response to the related pathogen A. cochlioides.
Plant glutathione S-transferases (GSTs) are ubiquitous and multifunctional enzymes whose expression is also induced under several form of abiotic and biotic stresses, including bacterial, fungal and viral infections [33]. They display a detoxification activity of toxic compounds by conjugating with glutathione, an oxidative stress attenuation and they participate in hormone transport [33]. In our study, the GO terms “detoxification” and “response to toxic substance” were enriched at certain levels in all samples at 6 hpi and 30 hpi and in response to different A. cochlioides isolates. The role of GSTs during infection by necrotrophic pathogens has been investigated in other pathosystems. For example, a proteomic study revealed an accumulation of catalase 3 and multiple GSTs in A. thaliana, following infection with the necrotrophic fungus Botrytis cinerea, demonstrating the importance of an antioxidant system in defense against the fungus [34]. Catalase 1 and GSTs are known to protect host cells against reactive oxygen species. The breakdown of hydrogen peroxide (H2O2) by conversion into molecular oxygen and water is mediated by catalase 1 [35]. Therefore, the detoxification activity that undergoes changes during infection with A. cochlioides could be a consequence of the accumulation of H2O2 formed during plant-pathogen interactions.
Germin-like proteins (GLPs) are ubiquitous glycoproteins reported in both higher and lower plants, characterized by several biochemical properties such as oxalate oxidase (OxO) and superoxide dismutase (SOD) activities [36]. They are known to be involved in plant defense to biotic and abiotic stresses and several studies have demonstrated their role in plant basal resistance to fungal pathogens [37]. GLPs also belong to the pathogenesis-related protein family and are part of plant basal resistance [38]. Many of them are localized to the plant cell wall and play a role in cell wall reinforcement by participating in the cross-linking of cell wall components [39]. The germin-like protein BvGLP-1 gene from sugar beet, which exhibits sequence similarity to other plant GLPs, is highly up-regulated in resistant plants during nematode infection. The over-expression of BvGLP-1 in A. thaliana constitutively activated the expression of a subset of plant defense-related proteins, inducing resistance against the pathogens Verticillium longisporum and R. solani, by elevating the levels of H2O2 [40]. A novel GLP isolated from cotton (Gossypium hirsutum L.), GhABP19, was demonstrated to be involved in the defense against the fungal pathogens V. dahliae and Fusarium oxysporum [41].
The increased expression of these genes in both partially resistant and susceptible lines indicates that they play an important role in constitutive defense responses to stress conditions and invading pathogens. However, the inhibition of the pathogen growth depends on the ability of the plant to recognize and identify the invader and its secreted molecules. Therefore, the pathogen could be able to overcome this first barrier of defense and to suppress downstream host defenses in the susceptible hosts.
Enriched GO biological processes and pathways in partially resistant and susceptible sugar beet genotypes in response to A. cochlioides
Changes in genes associated with the photosynthesis (i.e., the light reaction), following the infection were the most overrepresented in the two susceptible sugar beet lines, especially at 30 hpi. It is known that the photosynthesis is not only affected by changes in the environmental conditions and abiotic stresses but also by pathogen invasion [42]. However, the mechanisms that mediate these changes after pathogen attacks still remain unclear. A possible reason could be an increased demand for assimilates from the pathogen but changes in the photosynthesis and other assimilatory metabolisms are also proposed to be a plant strategy to invest energy in coping with the pathogen invasion [43]. Alterations in the photosynthesis, but in the dark reaction, were observed also in the partially resistant genotype G06 at 6 hpi, suggesting that this genotype might promptly re-direct energy to defense responses. KEGG analysis showed that several DEGs in the susceptible lines were part of photosynthetic pathways (i.e., carbon fixation, photosynthesis). In addition to changes in photosynthetic activities, which appeared to be predominant in genotype G01, other responses were triggered in genotype G17, which include genes with GO terms related to interspecies interactions and responses to fungus/another organism. Genes involved in defense responses were also up-regulated in G17 at 30 hpi, indicating that host defense responses are elicited in this genotype at the initial phase of the infection and are probably overcome in later stages by the pathogen.
The overall responses to A. cochlioides infection between the two partially resistant genotypes G06 and G12 appeared to be diverse. The most enriched GO terms in genotype G06 were associated to ROS metabolic process, in particular H2O2 and to detoxification and response to toxic substances. H2O2 is known to play multiple roles in plant responses to pathogens. It directly limits the viability and the spread of the pathogen, it induces the production of phytoalexins, it is involved in the localized plant cell death during the hypersensitive response, it acts as signal to the systemic acquired resistance and lastly it induces the expression of defense genes [44]. Changes in genes associated with the metabolism of H2O2 suggest that it might have a central role in regulating defense responses to A. cochlioides, while the enrichment of GO terms associated to detoxification could be interpreted as a consequence of the high accumulation of H2O2 which, at high levels, is toxic and can cause damage to cell structures [45]. Similar responses were observed in the susceptible genotype G17 at the earliest time point. These results might indicate that similar mechanisms are activated in response to the pathogen invasion in both the partially resistant and the susceptible genotypes, but the interaction with the pathogen subsequently leads to different reactions in the two hosts. In the other partially resistant genotype G12, the most interesting GO biological process was cell wall organization. The cell wall acts as physical barrier to prevent the pathogen ingress in the cell and, in response to an attack, plants can deposit polymers to strenghten the cell wall [46]. Changes in genes associated to H2O2 metabolism were observed also in genotype G12 at the earliest time point (6 hpi), confirming that during the infection process changes in the metabolism of this ROS are induced. H2O2 is also required for peroxidase-dependent lignification and for protein cross-linking in the cell wall, making the cell more resistant to cell wall-degrading enzymes [46]. Therefore, a cell wall reorganization could be a defense mechanism triggered in genotype G12 to attempt to physically prevent penetration by the pathogen. In addition, the GO biological processes “response to chitin”, “response to other organism”, “interspecies interaction” and “defense response” were enriched in genotype G12 at 30 hpi, indicating the induction of specific responses following the detection of the pathogen.
Differentially expressed genes involved in host plant resistance towards A. cochlioides
Resistance (R) proteins act as primary receptors of pathogen effectors or have an indirect effect in the recognition process [47] and initiate signal transduction pathways that result in the expression of disease resistance through activation of the hypersensitive response (HR) and other responses [48]. Among the up-regulated genes observed in the two partially resistant genotypes but not expressed in the susceptible lines, 8 genes were in common between G06 and G12. One of these genes corresponds to a gene annotated as “Probable disease resistance protein At1g15890”, that belongs to the CC-NBS_LRR class of disease resistance proteins. Another of these genes is annotated as mitogen-activated protein kinase 4 (MKK4), which is involved in the second phase of H2O2 generation during HR. In addition, in genotype G06 we observed the up-regulation expression at 30 hpi of genes annotated as homologues of the A. thaliana genes NDR1, which is linked to many R genes and is required for the establishment of both HR and systemic acquired resistance (SAR) [49],and the NBS-LRR resistance gene RPM1, that, for example, leads to the restriction of Pseudomonas syringae growth through HR [50]. An homologous gene of NDR1 was also up-regulated in a susceptible pea genotype in response to A. euteiches infection at 48 hpi [27] Three putative disease resistance proteins (RPP13-like protein 1, RGA3 and At3g14460) and the disease resistance protein RGA2 were up-regulated in genotype G12. The RPP13 resistance gene family was first discovered in A. thaliana and confers resistance to downy mildew caused by the oomycete Hyaloperonospora parasitica [51]. It functions independently of NDR1 and EDS1 and does not require the accumulation of salicylic acid for activation [52]. AtLRRAC1 (At3g14460) is a LRR class R gene with adenylyl cyclase activity which is cAMP dependent and promotes defenses against biotrophic and hemibiotrophic pathogens in A. thaliana but has not been shown to be active against necrotrophic pathogens. It is therefore hypothesized to be involved in early PAMP Triggered Immunity (PTI) signaling rather than acting as a classical R-gene that recognizes specific effectors [53]. Our results therefore suggest that the two genotypes might initiate an immune response via the so-called effector-triggered immunity (ETI), upon R genes recognition of A. cochlioides effector(s). However, more generalized, earlier acting PTI responses may also be important for resistance to A. cochlioides. Since it is now well known that these pathways are convergent and potentiate one another, [54] further research is required to untangle the precise timing and activation of these responses in sugar beet. Furthermore, multiple different pathways representing both NDR1 dependent and independent mechanisms may be activated within the defense responses of the different genotypes. It is possible that genotype G06 is dependent on NDR1 pathways, whilst G12 may use NDR1-independent pathways, although further research is required to test this hypothesis. In addition, an up-regulated gene annotated as MLP-like protein 43 was identified in genotype G12 at 30 hpi. Besides their role in drought and salt tolerance, Major Latex-like Proteins (MLPs) are also known to induce resistance against pathogens [55]. Three MLPs homologous genes have been reported to be highly expressed in sugar beet roots of partially resistant genotypes, 5 days after inoculation with the soil-borne basidiomycete R. solani [15]. Their role in plant defense has been observed also in response to other fungal pathogens, namely V. dahlia [56] and Alternaria brassicicola [57] and to the soil-borne plasmodiophorid Plasmodiophora brassicae [58]. Although their function remains to be investigated, the expression of these genes during the infection process indicates that they play an important role in regulating plant resistance mechanisms and, therefore, that the MLP-like protein 43 could contribute to defense responses to A. cochlioides in the sugar beet partially resistant genotype. Another gene annotated as pathogenesis-related protein (PR), NtpR, was expressed in genotype G12 at 30 hpi. A previous study where the NtPR-Q gene has been over-expressed in tobacco (Nicotiana tabacum L.) has shown the role of this gene in inducing expression of defense-related genes and in defending tobacco against the pathogen Ralstonia solanacearum.
In addition, several TFs were identified among the DEGs of the different genotypes. Some of them are known to be associated with plant defense responses. Among the up-regulated genes, we detected several ethylene-responsive TFs (ERF8, ERF095, ERF109 and RAP2-3) which bind the GCC-box promoter element of pathogenesis-related (PR) genes, whose expression is up-regulated following pathogen attack and WRKY40 and WRKY70, belonging to the WRKY family, which regulate the defense responses to bacteria, fungi and oomycetes. A TF belonging to the HB family, OCP3, was also detected in the partially resistant genotype G12. OCP3 has been reported to modulate NPR1-mediated jasmonic acid-induced defenses in A. thaliana [59].