Discussion
With the aim to explore biological processes involved in the mechanism of resistance/susceptibility of C. sativus against the oomycete,P. melonis , we chose to undertake an analysis of expression levels of five candidate defense-related genes in leaves and root collars of selected cucumber genotypes that possibly contribute to the establishment of damping-off resistance in the cucumber plant. Therefore, four cucumber genotypes, which according to the recent screening test results showed resistance (Ramezz), moderately resistance (Baby) and high susceptibility (Mini 6-23 and Extrem) to damping-off, were used to analyze the transcriptional activity of CsWRKY20, CsLecRK6.1, PR3 , PR-1a and LOX1 genes during infection produced by P. melonis. The higher activities of defense enzymes such as PAL and POX, and higher level of H2O2 in infected resistant Ramezz and moderately resistant Baby tissue compared to the high susceptible genotype Mini 6-23 and Extrem may explain theirs high level of resistance. The contrasting degrees of susceptibility of these cucumber genotypes to damping-off represents a good opportunity to investigate their specific molecular responses in order to better understand underlying mechanisms governing the resistance or susceptibility to P. melonis. This study revealed obvious similarity between leaves and root collars in their response to damping-off. Moreover, the transcripts of these five genes accumulated significantly at a greater level in Ramezz and Baby than in Mini 6-23 and Extrem upon inoculation with P. melonis and probably being involved in early responses against P. melonis .
There is a large number of evidence that suggests critical role of WRKY TFs in regulating defense related genes and increasing disease resistance to pathogens in several plant species (Alves et al. 2014). In addition, some WRKY TFs act as positive regulators of SA-dependent defense responses and positively regulate PR gene expression (Eulgem and Somssich 2007; Liu et al. 2018). In the current study, the expression of CsWRKY20 gene was induced after infection byP. melonis and a significant accumulation of CsWRKY20transcripts was observed in Ramezz and Baby in the present study. So the plants constitutively expressing CsWRKY20 were more resistant to damping-off. Our finding is in accordance with previous studies which have demonstrated the association of WRKY TFs in resistance response against oomycete phytopathogens. Xu et al. (2015) predicted thatCsWRKY20 may be involved in oomycete disease resistance of JSH (resistant cucumber cultivar) against P. melonis by JA and (or) SA signaling pathway(s). The WRKY40 positively regulated the expression of defense-related genes of chickpea and enhanced disease resistance against Pseudomonas syringae (Chakraborty et al. 2018). Another study has shown, the SpWRKY3 may enhance tomato resistance to tomato late blight (Phytophthora infestans ) as a positive regulator (Cui et al. 2018).  Therefore, this is not surprising considering the fact that CsWRKY20 in cucumber might has a similar role during interaction between cucumber and P. melonis. Further research through the development and analysis of loss of function mutants for CsWRKY20 gene is needed to clarify its role in cucumber signaling in response to P. melonis infection.
Previous study showed that CsLecRK6.1 may play an important role in disease resistant against P. capsici and P. melonis in JSH resistant cucumber (Wu et al. 2014). Additionally, multiple studies have been revealed the significant and outstanding role of LecRKsin plant innate immunity on several aspects that make them fascinating as potential resistance components in plant (Zhao et al. 2018). Accordingly, we selected CsLecRK6.1 as candidate and study results Demonstrate that CsLecRK6.1 responded to P. melonis in the Ramezz and Baby and a significant accumulation of gene transcripts was observed in these genotypes for all the time point after inoculation. Bouwmeester et al. (2011) demonstrated that expression ofLecRK-I.9 was induced by inoculating with non-host and avirulent pathogens in Arabidopsis and subsequently enhanced resistance toPhytophthora brassicae by overexpression of LecRK-I.9 . A recent study by Wang et al. (2018) showed that LecRK-V confers broad-spectrum resistance to powdery mildew in wheat and SA pathways contribute to the enhanced resistance to the powdery mildew fungus. However, the expression information of CsLecRK6.1 gene will be useful for further investigating the function of this gene under various biotic stress conditions.
We also focused on the expression pattern of some PR genes that are reliable defence markers in cucumber, such as PR1-1a, a marker of the SA pathway, PR3 encoding chitinases. PR proteins are accumulated in various parts of normal tissues and are induced by pathogen infection and improve the defensive capacity of plants. Members of the PR-1 and PR-3 families of proteins have direct activities against oomycete pathogens (Sudisha et al. 2012). Sterol-auxotroph pathogens such as the Phytophthora are particularly sensitive to PR-1 families and plants with enhanced PR-1 expression are particularly well protected against oomycete (Gamir et al., 2017; van den Berg et al. 2018). The transcripts of PR1-1a was significantly upregulated in both inoculated leaves and root collars of moderately resistant and resistant cucumber plants.
It is thought that Chitinase1, which is encoded by PR3 and can hydrolyze chitin, contributes to the defense of plants (Rawat et al. 2017). Study of cucumber and nonpathogenic F. oxysporum CS-20 interaction have shown a rapid response to pathogen inoculation at the initial stage, 24 hpi, and that it increased again at 48 hpi and then reduced again at 72 hpi (Pu et al. 2014). In this study, we find similar patterns of PR-3 expression. The significant and active accumulation of PR1-1a and PR-3 in Ramezz and Baby suggests a crucial role of these transcripts in direct defense mechanism of cucumber against the P. melonis . Therefore, it may be hypothesized that the difference in susceptibly between the four genotypes could be explained, at least partly, by the differential expression of genes involved in anti-oomycete defense.
In response to P. melonis , Ramezz and baby show greater levels of induction of lipoxygenase gene, LOX1 coding for enzyme of the biosynthesis of JA. Previous studies support the role of this gene in plant defense against oomycete and fungal infection (Rancé et al. 1998; Maschietto et al. 2015). Pathogen-induced LOX transcript accumulation has been reported in a number of plants, e.g. in cucumber after inoculation with F. oxysporum CS-20 (Pu et al. 2014), in tobacco after infection with P. parasitica var. nicotianae (Rancé et al. 1998) and in potato infected by P. infestans (Göbel et al. 2002). Although, this interaction is not the same, we found a similar profile in our experiment, in which LOX1 showed activation after inoculation by P. melonis . Consequently, the up-regulation ofPR1-1a and PR-3 on one side and the upregulation ofLOX1 on the other side, suggests a much intense activation of SA and JA pathways in Ramezz and Baby than in Mini 6-23 and Extrem, which probably promote the tolerance to P. melonis .
Our results indicated that CsWRKY20 and CsLecRK6.1 as well as PR genes (PR3, PR-1a ) responded quickly in leaves and root collars to P. melonis stress, suggesting that these responses might be central components of the induced immune system through JA and SA signaling pathway. surprisingly, among the 5 defence-related genes monitored in infected leaves and root collars, theLOX1 and CsLecRK6.1 gene were highly upregulated in the inoculated cucumber plants, respectively. In addition, the similar response of these genes expression may indicate that same control mechanisms of these genes may exist in leaves and root collars. Additionally, infection of cucumber with P. melonis resulted in a significant decrease in fresh and dry weights of roots and aerial parts, root volume and collar diameter, although the magnitude of these decrease depended upon the genotypes. Previous research showed that all Phytophthora species were able to cause damage to the root and shoot growth parameters (Aghighia et al., 2016; Ruiz Gómez et al., 2018).
Our results showed that the defense response due to P. melonisinoculation varied in resistance, moderately resistance and high susceptible genotypes of cucumber and the difference between resistance and susceptibility depends on early detection of the pathogen. Based on the fact that the expression of PR3 , PR1-1a , LOX1 ,CsLecRK6.1 and CsWRKY20 genes in infected plants was significantly different from the control at 0 hour before inoculation with P. melonis , it is suggested that these genes mostly participate in defensive response. Furthermore, these genes showed different levels of transcript accumulation in investigated genotypes and the higher levels of expression of these genes was observed in Ramezz and Baby. All these results strongly indicated that underP. melonis stress conditions leaves and root collars of different cucumber genotypes employed same mechanisms to cope with damping-off.
Although, this is a preliminary study on C. sativus - P. melonis interaction, but could be useful to understand defense mechanism of cucumber. Additionally, the study of these gene expression profiles will further promote the functional research of cucumber plant defense responses and signaling pathways in the future. However, more research is needed to detect the differences and gain an insight into the more detailed characteristics of these genes and interactions ofP. melonis effectors with the genes under study.