*2.6. INA Priming Prevented Enzymatic Digestibility of CWs*

The CW changes observed after the Pph inoculation, and especially after the INA priming, demonstrated CW remodeling events. In order to know whether such CW remodeling confers more resistance to pathogen attack, especially taking into account that Pph is able to produce CWDE, a time course of sugar release after enzymatic hydrolysis of crude CWs was carried out (Figure 9). In this assay, no significant differences were found when both, sugar yield and sugar release kinetics obtained from Mock + Pph and Mock CWs were compared. However, INA pre-treatments induced a slower release of total sugars during the first half of the time course. Samples from INA + Pph did not reach sugar release levels of Mock after 24 h. In summary, the INA priming induced a CW reinforcement, making the structure more resistant to enzymatic hydrolysis.

**Figure 9.** Time course of sugars released by enzymatic hydrolysis of CWs extracted from Mock, Mock + Pph, INA and INA + Pph conditions. Data represent mean ± SE (*n* = 3). Statistical analysis was carried out by two-way ANOVA where letters indicate significant differences by Tukey test (*p* < 0.05).

#### **3. Discussion**

In nature, plants must overcome multiple stresses, such as those induced by pathogens, by activating several defense mechanisms. The CW is the first plant defensive layer against intruders. Plants are able to rearrange this structure on demand at different developmental stages. The CW can be reinforce by modifying the proportions of its main components, and/or by changing the type and extent of cross-linking among CW polymers [4]. To gain insight into the pathogen and priming capacity to reinforce the CW, the remodeling of CWs after the Pph inoculation and INA priming in the Pph-susceptible common bean variety *Riñón* was studied in this work.

Previous studies have suggested the ability of INA, as a SA structural analogue, to trigger the immune system in common bean [40,42,49]. To confirm the priming activation in *Riñón* common bean, plants of this variety previously treated or not with INA were inoculated or not with Pph (Supplementary Figure S1). By following that approach, it was confirmed that plants pre-treated with INA showed less halo blight symptoms [45], as the chlorosis and greasy appearance observed after the Pph inoculation decreased (Figure 1). The fact that these symptoms were constrained to damage at the foliar margins confirmed that INA pretreatment prior to the Pph inoculation protected *Riñón* bean plants from halo blight disease, as it was previously observed in other varieties [42,49]. As priming suggests, this observation may indicate that cell defensive responses have already been activated, and consequently, a stronger defensive mechanism is expected [37,41]. In order to confirm such hypothesis, the ROS burst triggered by peptide flg22 [70] was evaluated in bean leaf disks previously incubated or not with INA. As shown in Figure 2, a more intense ROS signal after flg22 triggering was detected in those plant disks previously treated with INA, which was in line with the priming concept, as it primed the plant cells to develop a more vigorously defense response somehow [71–74]. Ramirez-Carrasco et al. (2017) [42] proposed that this SA analogue produces epigenomic changes in bean that can even be inherited [43,49].

The presence of a CW that shields plants from pathogen invasion is a common resistance mechanism to disease among all plant cells. Several recent works have demonstrated a more relevant role of the CW than initially expected mediating such resistance. Most of them have focused on the study of mutants with a great variety of CW alterations, which have resulted in differential resistance or susceptibility against plant pathogens [4,20]. Other works have studied the role of elicitation with conserved CW-derived molecules from the pathogen, or from the plant itself, which once perceived the pathogen and triggered different immune responses [75,76]. However, the knowledge about CW remodeling after immune priming or bacterium infection is scarce. Here, we studied whether Pph

inoculation and/or INA priming in common bean could promote a CW remodeling which would end up in the enhanced protection against halo blight disease, as shown in Figure 1.

The study of CW remodeling in this work began with the non-invasive technique FTIR spectroscopy of crude CWs, which resulted in the discrimination of FTIR spectra of Mock + Pph, INA and INA + Pph samples from the Mock samples (Figure 3). Dimension 2 of the PCA applied, which explained almost 30% of variance, was mainly related to wavenumbers associated with pectins, cellulose, and phenols (Table 1). The subsequent analysis of the polysaccharide content revealed changes in the amount of cellulose (Figure 4), matrix polysaccharide distribution (Figure 5), monosaccharide composition (Figure 6), and changes in the inmunoprofile of pectins (Figure 8).

When bean plants were inoculated with Pph, no statistical differences were found in cellulose content (Figure 4), in contrast with previous findings from other pathosystems [3]. However, our results point to both quantitative and qualitative changes in pectins and hemicelluloses after the Pph inoculation (Figures 5 and 6). The total amount of pectins (CDTA and Na2CO3 extracted polysaccharides) increased upon the Pph inoculation (Figure 5). Indeed, the higher HG methylation degree observed, as revealed by immunolabeling in these fractions (Figure 8), would suggest a HG synthesis increase after the infection, as HG methyl-esterification only occurs in Golgi apparatus [77–79], and/or a decrease of pectin methyl esterase activity after the Pph infection, as this is altered in other pathosystems [27,80]. Besides, the Ara and Xyl variation in the KII fraction suggest an increment of AX in tightly cross-linked hemicelluloses. The Mw of pectins (particularly those Na2CO3 extracted) and loosely crosslinked hemicelluloses (KI fraction) raised after the Pph inoculation. A putative increase in the length and/or number of arabinan and galactan side chains of RG-I was also reported.

Several studies show how plants modify their pectin methylation degree or substitution pattern in order to avoid microbe colonization [78,81]. It is well-described that HG demethylation, as it is necessary to establish links between HG chains through Ca2+ bonds to form the denominated egg-box complexes [82], participate in biotic resistance [30,31]. This type of link could explain the Mw increase observed for pectic fractions (Figure 7) [83,84]. These changes in pectic polymers could be not enough as defensive mechanisms, as it was observed phenotypically (Figure 1). In fact, the CW degradability profile was similar in Mock + Pph and Mock (Figure 9). Pph is characterized, as other pathogenic microbes, by the production of CWDE that are released in the apoplast, similar to those used in the CW degradability assay [4,48]. This could be an explanation to the symptoms observed caused by the disease.

The Glc increase in the TFA fraction may reflect the accumulation of non-crystalline cellulose after the INA treatment (Figure 4). INA, as structural SA analogue, could stimulate cellulose synthesis. This polysaccharide is precisely regulated by growth factors, and previous studies have suggested the relation between SA and cellulose accumulation [85].

Alternative explanations for the Glc increase in the TFA fraction would be the accumulation of callose and/or the enrichment in a XG population tightly bound to cellulose. Both explanations seem plausible as the protective role of callose against Pph attack [86] and the function of XG by strengthening CW structure [63,64] have been previously reported. The CW fractionation of plants pre-treated with INA showed a noticeable displacement of matrix polysaccharides among fractions.

Regarding Mock and Mock + Pph conditions, neutral sugars were barely detected in CDTA and Na2CO3, decreased in SnCR, and increased in TFA (Figure 5). Specifically, the displacement of monosaccharides to the TFA fraction would suggest that RG-I, XG and xylans are the polysaccharides involved (Figure 6). A similar displacement from CDTA to Na2CO3, KII and SnCR could have occurred regarding uronic acids (Figure 5B). These changes indicated that polysaccharides become strongly attached to the matrix. Moreover, marked changes in polysaccharide size (Figure 7) and inmunoprofile in epitopes for each fraction (Figure 8) would suggest INA-dependent changes in the CW structure. The increase in glycan Mw observed for all fractions in INA treatments compared to

Mock (Figure 7) could be explained by changes in cross-linking detected in CW epitopes (Figure 8), which is interesting when the sugar content decreased in CDTA and did not change in KI and KII (Figure 5). Galactan or arabinan side chain substitutions in RG-I decreased in CDTA but increased in Na2CO3 after the INA pretreatments. Additionally, AGPs or XG diminished after the priming, but all fractions obtained from plants pre-treated with INA showed an increment in HG and extensin epitopes (Figure 8). As being part of the same molecule [87,88], the changes observed in RG-I could have indirectly rendered a HG more attached to the matrix and, consequently, the detection of HG in fractions such as KI and KII, in which the presence of pectins is not usually abundant [89,90].

The more distinctive fact of CW fractions from INA treatments compared with those obtained from Mock and Mock + Pph CWs, was the increment in extensin epitopes, which could participate in matrix remodeling, maintaining the plasticity. Extensins are hydroxyproline-rich glycoproteins which interact with other CW components such as pectins, participating in the architecture, the structural organization and the strengthening of the CW [91–93]. Therefore, these proteins are involved in polysaccharide crosslinking [92,93]. Interestingly, extensin overexpression produces a decrease in the susceptibility of Arabidopsis to *Pseudomonas syringae* DC3000 [94]. In line with this, Arabidopsis plants with a reduced wall-extensin content are less resistant to *Botrytis* [26]. In addition, it is well known that some extensin genes are induced in *Arabidopsis* and *Nicotiana* by SA [95–97], and INA, as a SA analogue, could produce a similar effect in bean.

Taken together, all these changes suggest a remodeling of the CW architecture, which was finally confirmed by the enzymatic digestion assays, as CWs extracted from plants treated with INA showed a higher resistance to enzymatic hydrolysis (Figure 9). These differences, which were especially noticeable at early times, could be explained, at least partially, by a CW reinforcement after priming. The inoculation of the pathogen after the INA priming meant slight CW modifications, which was also reflected in a similar CW degrading profile upon enzymatic digestion. As previously reported [98], the CW cross-linking and strengthening could hamper pathogen CWDEs activity and confer biotic resistance, which would explain the slight lesions produced by halo blight after the INA pre-treatment in this variety (Figure 1). INA pre-treated plants were more resistant to Pph, and showed small necrotic spots, which resembled those produced during the HR response (Figure 1D). In resistant plant species, SA accumulates in the infection site at early stages of the immune response, which leads to HR [99]. However, previous studies showed that *Riñón* variety was not able to produce the early SA peak required after the Pph attack to induce HR [47]. Therefore, INA could replace the role of SA by increasing the defense signal at early stages after Pph infection in this variety.

To summarize, the Pph inoculation modified the common bean CW to a much lower extent than INA-priming. It did not involve an increase in cellulose content, but increased pectins (HG and RG) in pectic fractions and hemicelluloses (AX) in KII fraction. The Mw of polysaccharides increased most of all in Na2CO3 fraction, but the CW was as vulnerable to enzymatic hydrolysis as Mock. By contrast, INA priming triggered a drastic CW remodeling, by increasing the cellulose content, displacing the matrix polysaccharides among fractions, and increasing the Mw of polysaccharides extracted in each fraction. This could be related to an increment in pectins (HG and RG) and extensins in all fractions, which supported a more extensive cross-linking that resulted in a CW more recalcitrant to enzymatic hydrolysis. The Pph inoculation after INA priming did not modify substantially this CW remodeling and the resultant CW was as resistant to enzymatic degradation as INA without inoculation. Therefore, INA-priming-phenotypes regarding more intense ROS production after flg22-elicitation and increased resistance against Pph infection, could be explained, at least partially, by the CW remodeling observed. Future work to unveil the specific links between CW remodeling and disease resistance will pave the way to design novel crop protection strategies based on such knowledge.
