**1. Introduction**

*Erwinia amylovora* causes fire blight, a disease of agronomic and economic importance that affects many Rosaceae species, primarily pear and apple trees. Bacteria penetrate the plant mainly through the flowers and can also enter leaf tissue through wounds [1]. During plant-pathogen interaction, a dialogue occurs between the two organisms: the

**Citation:** Sgamma, T.; Forgione, I.; Luziatelli, F.; Iacona, C.; Mancinelli, R.; Thomas, B.; Ruzzi, M.; Muleo, R. Monochromic Radiations Provided by Light Emitted Diode (LED) Modulate Infection and Defense Response to Fire Blight in Pear Trees. *Plants* **2021**, *10*, 1886. https://doi.org/ 10.3390/plants10091886

Academic Editor: Magda Pál

Received: 24 August 2021 Accepted: 8 September 2021 Published: 12 September 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

plant synthesizes molecules for the signaling system and defense; in contrast, the pathogen synthesizes molecules to break down the barriers of the host and mimic plant hormones. In the beginning, under favorable climatic conditions, the pathogen inserts through the intercellular spaces of parenchyma. Afterward, it colonizes the xylem vessels causing extensive damage with the final death of the plant [2]. The necrotic parts of the plant become brown as if they had been burned by fire [3].

During the infection process, pathogenesis-related (PR) proteins are part of an articulated systemic signaling network active in plants to perceive pathogens and activate defenses. The necrotic lesion induces the expression of a set of pathogenesis-related genes. *PR1* gene, one of the 17 *PR* gene families, has frequently been used as a marker for systemic acquired resistance (SAR) in many plant species [4–6]. In the immunization stage, necrosis-causing pathogen when infects a leaf usually provokes the formation of a localized dry, necrotic lesion that limits pathogen spread and provides local resistance. This step is also referred to as hypersensitive response (HR) [7]. Accumulation of salicylic acid (SA) is also associated with this stage. SA is an endogenous plant hormone whose levels increase after pathogen infection. SA can induce the expression of *PR1* [8]. Increases in SA and SA-inducible PR proteins are associated with disease resistance at several levels, not just with the SAR response. A phloem-mobile signal then moves from the immunized leaf to the rest of the plant to establish SAR. The perception of the mobile signal in the uninoculated leaves results in the expression of the same set of *PR* genes as induced around the primary infection site. When the plant is challenged with a second virulent pathogen, the plant responds as if that was an avirulent one because of the rapid accumulation of the *PR1*-transcripts [9]. However, there are indications that several of the *PR* genes are expressed at basal levels in plants without any pathogen attack. Moreover, studies showed that SA is also crucial for sustaining basal levels of genes associated with resistance responses, including *PR1*, and keeping the defense system primed in the absence of pathogen attacks [4,10–13]. Remarkably, when *PR* genes are not expressed this leads to a higher susceptibility to infectious agents [5].

The PR10s defense-related proteins are a ubiquitous class of intracellular in contrast to the extracellular nature of most PR proteins [14]. Most of them are induced upon microbial attack by fungal elicitors, wounding, and stress stimuli, as with most of the other PR-protein families. PR10 proteins are also expressed in a tissue-specific manner during development and some PR10 proteins show constitutive expression patterns [15,16]. PR10s have been attributed a ribonuclease-like function due to sequence homology with ribonucleases (RNase) [17]. However, only some PR10 proteins have been proposed to possess RNase activity [18]. In addition, they have also been shown to respond to plant hormones, including jasmonic acid (JA), and abiotic stresses such as salt and drought [18].

Plant defense responses at the site of the bacterial infection are elevated, accumulation of SA occurs and the transcription of *PR1* is induced in light [19]. The PR1 light dependency and the execution of HR confirm that these responses are closely associated and that light regulation already takes place early in this SA-dependent signaling pathway [7]. Phytochromes are crucial photoreceptors and are involved in the modulation of the *PR1* expression by light. The absence of both PHYA and PHYB strongly reduces the expression of *PR* genes upon treatment with SA, with a more significant influence of PHYB deficiency [5,7,20]. Phytochrome signaling strongly modulates the response of endogenous SA [5]. There is a strict light dependency of gene expression of *PRs* and the HR process. HR lesions are often correlated with the induction of *PR* genes and are also light modulated. HR is strongly reduced by the absence of phytochromes and amplified in an SA-dependent manner in the *psi2* mutant [5].

Moreover, photoreceptor proteins such as cryptochromes (CRYs), which are Blue light (BL) photoreceptors homologous to photolyases, seem to be involved in pathogen response. Proteomics study identified proteins with altered expression related to defense, stress, and detoxification in *cry1* mutant [21]. CRY1 positively regulates SAR, indeed, in *Arabidopsis*, the inactivation of the *CRY1* gene has a mild influence on the SA accumulation

and determines a reduction of the *PR1* expression; in contrast, the overexpression of this gene *CRY1* significantly enhances the expression of *PR1* [22]. Furthermore, other studies showed that in mutants of COP1 (constitutive photomorphogenesis 1), COP9, and DET1 (De-etiolated 1), which are part of the CRY1 signaling pathway, *PR* genes were highly up-regulated [23,24].

Prokaryotes have evolved a repertoire of photosensory proteins that determine changes in the external light and regulate cell physiology in a light-dependent manner [25]. Bacterial photoreceptors include proteins with a bilin-type chromophore (bacteriophytochromes) for sensing red light (RL) and far-red light (FRL) [26]. Moreover, they include proteins with photosensory domains for BL such as BLUF (BL sensing using flavin adenine dinucleotide [FAD]), LOV (light, oxygen, or voltage), PYP (photoactive yellow protein), and cryptochrome/photolyase (Cry/PHR) superfamilies, green- or blue-light-absorbing microbial rhodopsin [27,28]. In plant-associated bacteria, the number of candidate photoreceptors varies: *Pseudomonas syringae* pv *syringae* B728a and *Pseudomonas syringae* pv *tomato* DC3000 have two bacteriophytochromes (BphP1 and BphP2) and one LOV domain-containing histidine kinase (LOV-HK) [29]; anoxygenic phototrophs, such as Methylobacteria, can contain between 3 and 16 photosensory proteins [30].

In the Enterobacteriaceae, there is only one report indicating the presence of a *bphP* gene in *Enterobacter cloacae* [31]. At the same time, several studies show that these bacteria are sensitive to irradiation treatments with wavelengths in the range of visible, violet, and blue light [32–34]. So far, no gene encoding photosensory protein has been identified yet in the plant pathogenic Enterobacteriaceae species *E. amylovora*, and there is no evidence that the growth, phenotype, or virulence of this pathogen is affected by the light.

In this work, four *E. amylovora* genes (*erw*) were isolated that could be used as a marker to monitor the initial phase of the infection in asymptomatic plants. To investigate if the circadian internal clock, the light quality, and the related photoreceptors autonomously regulate the abundance of *PR1* and *PR10* transcripts, in vitro-cultured plantlets of Iranian pear cultivar *Dar Gazi-wild type* (*wt*), *Dar Gazi-phyB* (transgenic plant overexpressing *Arabidopsis phyB*, and *Dar Gazi-cry1* (transgenic plant overexpressing tomato *CRY1*) were exposed to different circadian experimental conditions, and continuous BL, RL, and FRL, emitted by light-emitting diodes (LED).

Transcriptional changes in host and pathogen gene expression during early *E. amylovora* infection indicated that both plant *PR*s and bacterial *erw* genes were temporarily expressed and differentially regulated. The results reported in this work indicate that photoreceptormediated signals regulate the expression of specific plant and pathogen genes in pear plantlets infected by *E. amylovora*.
