## 2 Results and Discussion
### Effect of Salinity and Rhizobacteria on the Expression of BnRSH Genes  
Soil salinity stress mitigates crop productivity and is an important challenge for global sustainable agriculture [64]. It affects several aspects of plant metabolism leading to significant decreases in plant growth and yield [6]. _B. napus_ is considered one of the most saline-resistant species in the genus _Brassica_, being more tolerant not only than its diploid ancestors, but also than other polyploid species [65]. Salinity had a visible impact on _B. napus_ seed germination (Supplementary Figure S5) and the growth of 6-day-old rapeseed seedlings (Supplementary Figure S6). The germination ratio was visibly decreased even in 50 mM NaCl whereas in the presence of 200 mM NaCl less than half of the seeds germinated in comparison to the control (seeds germinated in water). The length of root and hypocotyl, as well as the fresh and dry biomass of _B. napus_ seedlings, significantly decreased in the presence of salt (Supplementary Figure S6) and the most affected by NaCl was hypocotyl growth (Supplementary Table S3). The potential involvement of _RSH_ genes and alarmones in the plant response to salt stress has been shown previously [26; 43; 44; 60]. In order to gain more insight into the possible physiological roles of _BnRSHs_, the expression of four selected _B. napus_\(RSH\) genes (_RSH1\(b\), _RSH2\(b\), _RSH3\(a\), _CRSH_) was analysed using sqRT-PCR in seedling organs  
Figure 6: Frequencies of putative _cis_-regulatory elements in _B. napus_\(RSH\) genes (upper pie chart), and in _BnRSH1_, _BnRSH2/3_, and _CRSH_ genes (lower pie charts). Pie charts depict the _cis_-regulatory elements categorized in seven types according to their predicted functions. (Figure 7) in response to salt stress, and in response to the presence of PGPR bacteria (Figure 8). _BnRSH_ genes were differentially expressed in cotyledons and roots, i.e., _BnRSH1_ and _BnRSH2_ genes were highly expressed, while _BnRSH3_ and _BnCRSH_ mRNAs were expressed at a lower level in both organs. Using bistochemical staining of GUS activity, it was shown that in _Arabidopsis_, _AtRSH1_ and _AtRSH3_ were highly expressed in hypocotyls and leaves, whereas _AtRSH2_ and _AtCRSH_ were expressed in leaves. In the roots of seedlings only _AtRSH2_ was expressed, whereas in the roots of mature plants, _AtRSH3_ was also expressed [26]. Using RT-PCR, high expression of _AtRSH1_ and _AtRSH3_, and low expression of _AtRSH2_ in shoots, were also shown. In the roots, _AtRSH2_ and _AtRSH3_ were highly expressed, whereas _AtRSH1_ was expressed at a low level. _AtCRSH_ was not tested in this study [66]. In rice, _OSCRSH_ was expressed both in roots and shoots, however, in roots at a lower level than in shoots [31]. In contrast, in the cotyledons of _I. nil_ seedlings, _RSH1_, _RSH2_, and _CRSH_ were equally highly expressed, whereas in roots, _RSH2_ was highly expressed, _RSH1_ was expressed at the low level, and no expression of _CRSH_ was detected [25]. In general, salinity stress had no significant effect on the expression of _BnRSH_ genes in cotyledons and roots (Figure 8). The levels of _BnRSH2_ and _BnCRSH_ transcripts in cotyledons, and the levels of _BnRSH1_ and _BnCRSH_ in roots, slightly increased under salinity stress as compared with control plants, and the differences were statistically significant (Supplementary Tables S5, S6, S10 and S11). Interestingly, previous studies showed that _A. thaliana_ treated with 250 mM NaCl exhibited increased _AtRSH2_ expression, but that salt had no impact on the expression of _AtRSH1_, _AtRSH3_, and _AtCRSH_[26], whereas, in another study, treatment with 250 mM NaCl significantly increased both _AtRSH2_ and _AtRSH3_ transcript levels, decreased the amount of _AtCRSH_ mRNA, and had no impact on _AtRSH1_ expression [57]. Similarly, [PERSON] et al. [25] showed that salt stress (300 mM NaCl) elevated the _InRSH2_ transcript level, whereas both _InRSH1_ and _InCRSH_ did not show substantial changes in 5-day-old _I. nil_ seedlings.