*2.6. PR1 and PR10 Expression in Dar Gazi-cry1 and Dar Gazi-phyB in RL, FRL, and BL*

Studying the role of photoreceptors in the regulation of the expression of the PRs, phytochrome has a pivotal role in regulating the internal clock and the perception of the photoperiod. The expression level of the *PR1* gene in plantlets *Dar Gazi-phyB* exposed to continuous RL increases to the highest rate (Figure 10a and Figure S3). Moreover, the expression of this gene shows an oscillating trend. On the other hand, in the tissue of *Dar Gazi-wt* and *Dar Gazi-cry1* plantlets, the transcript level was constant, at a very low level of expression. Therefore, the photoconversion of phytochrome from the inactive (Pr) to the active form (Pfr) should play a permissive role (Figure 10a,b). *Plants* **2021**, *10*, x FOR PEER REVIEW 22 of 23

**Figure 10.** *PR1* expression in plantlets of *Dar Gazi-wt*, *Dar Gazi-phyB*, and *Dar Gazi-cry1*: (**a**) in 24 h continuous RL, panel; (**b**) in 24 h continuous FRL, panel; (**c**) in 24 h continuous BL, panel. Results are presented after normalization with *ef1A*. Data shown are the average of two biological replicates run in triplicate, with error bars representing SD. At each time point values with different letters significantly differ according to the analysis of the variance (ANOVA) and least significant difference (LSD) tests (*p*  ≤  0.05). **Figure 10.** *PR1* expression in plantlets of *Dar Gazi-wt*, *Dar Gazi-phyB*, and *Dar Gazi-cry1*: (**a**) in 24 h continuous RL, panel; (**b**) in 24 h continuous FRL, panel; (**c**) in 24 h continuous BL, panel. Results are presented after normalization with *ef1A*. Data shown are the average of two biological replicates run in triplicate, with error bars representing SD. At each time point values with different letters significantly differ according to the analysis of the variance (ANOVA) and least significant difference (LSD) tests (*p* ≤ 0.05).


The regulation of *PR10* expression under continuous RL was very similar in *Dar Gazi*

that show a different behavior, but are analogous between themselves (Figure 11b). The rate of gene expression observed in *Dar Gazi-cry1* indicates that the presence of CRY1 is required for the upregulation of this gene. An analogous oscillatory behavior appears in plantlets of both transgenic lines when Under continuous BL conditions (Figure 10c), the highest level of *PR1* expression in *Dar Gazi-cry1* plantlets was reached after 6 h of exposure to light. An oscillatory behavior appeared in plantlets of *Dar Gazi-wt*, while in plantlets of *Dar Gazi-phyB* a very low expression rate without any oscillatory behavior was observed.

exposed to continuous BL (Figure 11c), while in *Dar Gazi-wt* not oscillatory behavior was observed. Comparing the rate of the *PR10* gene expression of the plantlets of *Dar Gazicry1* under FRL with BL, it is surprising that the behavior was not the same. The hypothesis could eventually explain this divergent behavior, that the gene expression's promoting role is mainly regulated by phytochrome, and only partly co-regulate by cryptochromes. The regulation of *PR10* expression under continuous RL was very similar in *Dar Gazi* -wt and *Dar Gazi-cry1* plantlets (Figure 11a). An oscillatory transcriptional behavior was observed in both lines of plantlets, although when this behavior is compared to photoperiodic conditions (Figure 11a). In the *Dar Gazi-phyB* plantlets, the trend of oscillatory behavior is different In particular, after the 6th hour, an autonomous behavior was observed (Figure 11a).

*Plants* **2021**, *10*, x FOR PEER REVIEW 22 of 23

**Figure 11.** *PR10* expression in *Dar Gazi-wt*, *Dar Gazi-phyB*, and *Dar Gazi-cry1*: (**a**) in 24 h continuous RL, panel; (**b**) in 24 hh continuous FRL, panel; (c) in 24 h continuous BL, panel. Results are presented after normalization with *ef1A*. Data shown are the average of two biological replicates run in triplicate, with error bars representing SD. At each time point values with different letters significantly differ according to the analysis of the variance (ANOVA) and least significant difference (LSD) tests (*p*  ≤  0.05). **Figure 11.** *PR10* expression in *Dar Gazi-wt*, *Dar Gazi-phyB*, and *Dar Gazi-cry1*: (**a**) in 24 h continuous RL, panel; (**b**) in 24 hh continuous FRL, panel; (**c**) in 24 h continuous BL, panel. Results are presented after normalization with *ef1A*. Data shown are the average of two biological replicates run in triplicate, with error bars representing SD. At each time point values with different letters significantly differ according to the analysis of the variance (ANOVA) and least significant difference (LSD) tests (*p* ≤ 0.05).

*2.7. CRY1 Overexpressing Line Is More Resistant to Fire Blight*

Plantlets were observed for 96 h after the inoculation of *E. amylovora* to detect necrotic tissues. Necrosis symptoms appeared only in the shoot apex of *Dar Gazi-wt* plantlets after 36 h from the inoculation (Figure 12). After 96 h, the progress of necrosis that affected the entire stem was visible. In the plantlets of *Dar Gazi-phyB*, necrosis was detected in several leaf nodes throughout the stem only after 48 h from the inoculation (Figure 12). Surprisingly, *Dar Gazi-cry1* plantlets better tolerated the pathogen infection showed necrotic tissues after 72/96 h (Figure 12). When exposed to continuous FRL, the transcriptional profile of *PR10* in plantlets of *Dar Gazi-wt* resemble an oscillatory behavior analogous to that observed in photoperiodic conditions (Figure 11b). This is not the case for the plantlets of the two transgenic lines, that show a different behavior, but are analogous between themselves (Figure 11b). The rate of gene expression observed in *Dar Gazi-cry1* indicates that the presence of CRY1 is required for the upregulation of this gene.

An analogous oscillatory behavior appears in plantlets of both transgenic lines when exposed to continuous BL (Figure 11c), while in *Dar Gazi-wt* not oscillatory behavior was observed. Comparing the rate of the *PR10* gene expression of the plantlets of *Dar Gazi-cry1* under FRL with BL, it is surprising that the behavior was not the same. The hypothesis could eventually explain this divergent behavior, that the gene expression's promoting role is mainly regulated by phytochrome, and only partly co-regulate by cryptochromes.
