*3.3. Cryptochrome Increase the Defense against the Attack of E. amylovora*

In this paper, four *E. amylovora* genes were identified that can be used to monitor the diffusion of the pathogen in pear vascular tissues during asymptomatic and symptomatic periods (Figure 3). Quantitative expression analyses revealed several interesting features: the expression of the *erw* genes was modulated during the internal movement of the pathogen through the plant vascular system (Figure 3); the activation of these genes occurred at specific times during the infection, the temporal expression pattern was dependent upon the pear genotype (Figure 13). In both *Dar Gazi* transgenic lines, the expression of *erw2*, *erw3* and *erw4* was advanced by 12 h compared to *Dar Gazi-wt*, from 24 h to 12 h for *erw2*; 36 h to 24 h for *erw3* and *erw4* (Figure 13). Twelve h after the infection, the transcript levels of *erw1* in *Dar Gazi-wt* and *Dar Gazi-phyB* were similar and increased in the same proportion between 12 h and 24 h (Figure 13). These data suggested that, in infected *Dar Gazi-wt* and *Dar Gazi-phyB* tissues, the growth pattern and the number of *E. amylovora* cells per plant mass unit were comparable. The early activation of *erw2* and the increased *erw2*/*erw1* ratio at 24 h in *Dar Gazi-phyB* vs. *Dar Gazi-wt* were dependent on the overexpression of *PHYB* in the transgenic line (Figure 13).

In contrast, 12 h after the infection, the mRNA expression level of *erw1* in the transgenic *Dar Gazi-cry1* line was about two-fold higher than in *Dar Gazi-wt*, and this difference remained constant up to 48 h (Figure 13). These data indicated that the overexpression of *CRY1* stimulated *E. amylovora* growth in pear tissues and altered the expression of the other erw genes. Noteworthy, in *Dar Gazi-phyB*, the expression of *erw2* and *erw4* remained constant between 12 h and 48 h (Figure 13). At 48 h, there was no significant difference in the expression levels of the four *erw* genes between *Dar Gazi-wt* and *Dar Gazi-phyB* (Figure 13). In contrast, in the *Dar Gazi-cry1* line, the transcript levels of *erw1*, *erw2*, and *erw3* significantly increased between 36 h and 48 h, reaching the maximum relative abundance. In comparison, the expression of *erw4* had a maximum at 36 h and

decreased between 36 h and 48 h (Figure 13). These data indicated that the plant-pathogen interactions occurring during the pathogen invasion were differentially affected by the alterations of the phytochrome- and cryptochrome-modulated signals resulting from the overexpression of *PHYB* and *CRY1*.

In the pear tissue, pathogen invasion generated oxidative stress in the central cylinder and the cortex accompanied by a widespread disruption of the plasma membrane that developed from the basal portion to the apex of the plantlets (Figure 2a). In *Dar Gazi-wt*, the necrosis symptoms appeared 12–36 h earlier than in transgenic lines (Figure 12).

Interestingly, in the plantlets over-expressing the photoreceptors, the transcript levels of *PR1* and *PR10* were higher than in *Dar Gazi-wt* (Figure 13). Independently from the pathogen load estimated by the *erw* genes expression data, it should be noted that there was a correlation between the PR transcript level and the appearance of necrosis symptoms. Delayed symptoms occurred in the *Dar Gazi* lines, such as *Dar Gazi-cry1* (Figure 12), in which higher *PR1* and *PR10* transcription levels were observed (Figure 13). It was demonstrated that a wide range of endogenous and exogenous (a)biotic factors, including pathogen attack, accumulation of salicylic acid, and abiotic stress, can regulate temporally and spatially the expression of *PR* genes [53,54] and the secretion and accumulation of the corresponding proteins in the apoplastic space or the vacuoles [55]. The results of this work demonstrate that the accumulation of the PR proteins can interfere with the dynamic interaction occurring during the *E. amylovora* invasion and delay the infection of the pear host tissues.

It is known that the protein product of the Far-red Insensitive 219/Jasmonate Resistant1 (FIN219/JAR1) functions as a jasmonic acid (JA)-conjugating enzyme responsible for the synthesis of the Jasmonic Acid-isoleucine (JA-Ile), the physiologically active form [56]. Under BL, FIN219 plays a role in the regulation of phenotype development and bacterial resistance [57,58] and how it occurs under FRL, it interacts with CONSTITUTIVE PHOTO-MORPHOGENIC 1 (COP1), down-regulating also the levels of *COP1* and up-regulating the levels of *HY5* [59]. COP1 is involved in the negative control of nitrate reductase activity in Arabidopsis *cop1* mutant, reducing the availability of nitrogen [60]. The availability of nitrogen resulted responsible for both *Arabidopsis* resistance to *E. amylovora*, i.e., under nitrogen limitation, the resistance decreased due to the lower apoplastic reactive oxygen species (ROS) accumulation and increased expression of *E. amylovora hrps* genes [61,62]. Moreover, cryptochromes may work together with phytochromes to modulate plant defense responses. In *Arabidopsis*, CRY1 positively regulates the inducible resistance to *P. syringae* pv. *tomato*. The local resistance is down-regulated in the *cry1* mutant; in contrast, in plants overexpressing *CRY1*, the *PR1* gene expression is enhanced, and the resistance is significantly up-regulated [22]. These results agree with the increased expression level observed in *Dar Gazi-cry1* compared to in *Dar Gazi-wt*, where a significant increase of expression was already detected at 12 h from inoculation, for both *PR1* and *PR10* genes.

Although, many key molecular factors involved in the plant-pathogen interaction, from the plant perception of the pathogen (P/MAMPs, PRRs) to the activation of the PAMP-triggered immunity (PTI), and the Effector triggered immunity (ETI), are already known [63,64], in *E. amylovora*-infected plants, the regulation of the photoreceptors by the interaction with the major phytohormones, SA, JA, and ethylene remains to be explored.
