*2.2. Pph Inoculation and INA Priming Produce Different CW Fingerprinting Than That Observed in the Mock*

Several recent evidence support a more prominent role of the CW in plant immunity than previously believed, although the knowledge about plant CW remodeling after pathogen infection and/or immune priming is scarce [4,5,22,26]. With this in mind, CWs from Mock, Mock + Pph, INA and INA + Pph bean plants were extracted. Characterization of whole CWs began with the non-invasive technique FTIR-spectroscopy, used to obtain global fingerprints of the CW [54]. The FTIR spectra obtained were clustered in a Principal Component Analyses (PCA), which separated the treatments in two groups (Mock and the rest of the treatments) according to dimension 2, which explained 29.43% of variance. In order to predict the putative CW components contributing to such segregation, the wavenumbers whose values had a higher correlation with dimension 2 were extracted and summarized in Table 1 together with their associated CW components. Several wavenumbers were associated with RG-I, galactan and xyloglycan, (1148 and 1152 cm<sup>−</sup>1), pectins (1232 and 1244 cm−1), uronic acids (1616–1628 cm−1), or with arabinogalactans (1156 cm−1). In addition, the wavenumbers related to cellulose (1160 and 1164 cm−1) and phenolics (1632 and 1720 cm−1) were also found. Next, difference spectra against mock were calculated for each condition (Figure 3B). As a result, the profile between 1800 and 1170 cm−<sup>1</sup> approximately, which referred among others to wavenumbers regarding uronic acids and pectins, was similar among all conditions. By contrast, the region from 1170 to 800 cm−<sup>1</sup> associated to RG-I, Galactan, Xyloglycan, Arabinogalactan and Cellulose (Figure 3B, Table 1) only changed after INA pre-treatment (with or without further Pph infection) when compared to mock.

**Figure 3.** Cell wall fingerprinting. (**A**) Principal Component Analysis obtained from FTIR spectra of crude cell walls extracted from leaves of Mock, Mock + Pph, INA, and INA + Pph common bean plants. Each dot represents a biological replicate obtained in each of the 3 independent experiments carried out. PCA is plotted with dimensions 1 and 2, which explained the 43.36% and 29.43% of the total variance, respectively. (**B**) Average FTIR difference spectra obtained by digital subtraction of the Mock CW spectra from the CW spectra of the other treatments.


**Table 1.** FTIR wavenumbers (cm<sup>−</sup>1) with highest contribution to explain Dimension 2 as Cos2 Dim2 (Figure 1) and the CW component to which they are associated.

*2.3. INA Priming Induced Quantitative Changes in CW Polysaccharides Not Observed after the Pph Inoculation*

To deepen into the CW changes after the Pph inoculation and the INA priming, the cellulose content was measured in crude CWs. It should be taken into account that cellulose, a major CW component, has been described to participate in defense and remodeling [20]. Besides, our results indicate that cellulose contributes to discriminate among treatments after PCA of FTIR CW spectra (Table 1). Updegraff methodology confirmed that the INA pre-treatment resulted in an increased crystalline cellulose content in both INA and INA + Pph plants, which could indicate a CW reinforcement after priming, due to the fact that the simple Pph inoculation of Mock did not alter the cellulose content significantly (Figure 4).

**Figure 4.** Cellulose content (μg per mg of dry weight CW) of the indicated treatments. Data represent mean ± SE (*n* = 3). Statistical analysis was carried out by one-way ANOVA where letters indicate differences by Tukey test (*p* < 0.05).

In order to explore the possibility of a CW reinforcement occurring after INA priming, a sequential CW extraction of matrix polysaccharides was performed. During the CW fractionation, pectic polysaccharides are commonly extracted in CDTA and Na2CO3 fractions, while hemicelluloses are mainly extracted by incubation with different alkali (normally

KOH or NaOH) concentrations, which extract hemicelluloses slightly (KI fraction) and tightly (KII fraction) bound to cellulose and/or cross-linked (Cosgrove, 2018). The following fractions recover polysaccharides tightly bound to cellulose (SnCR) and amorphous cellulose (TFA) [61], respectively.

The Pph inoculation only had an effect in pectic fractions, producing an increment of neutral sugars and uronic acids in CDTA fraction as well as an increment of uronic acids in Na2CO3 fraction compared to mock (Figure 5). However, the INA pretreatment produced dramatic changes in the neutral sugar distribution, by decreasing its content in CDTA, Na2CO3 and SnCR fractions, and significantly increasing it in TFA fraction (Figure 5A). The uronic acid content diminished in CDTA by INA pretreatment, but increased in pectic (Na2CO3) and hemicellulosic (KII) fractions, as well as in the SnCR fraction (Figure 5B). These results show a clear shift of polysaccharides from the CDTA fraction to others where extracted polymers were linked to the matrix more strongly, thus suggesting changes in their composition and/or structure. Interestingly, these CW changes observed in INA were similarly observed after the Pph inoculation (INA + Pph).

**Figure 5.** Biochemical composition of CW fractions from Mock, Mock + Pph, INA and INA + Pph inoculated common bean plants: (**A**) neutral sugars and (**B**) uronic acid content. Data represent mean ± SE (*n* = 3). Statistical analysis was carried out by one-way ANOVA where letters indicate differences by Tukey test (*p* < 0.05).

Thereafter, the fractions were subjected to acid hydrolysis, and neutral sugar composition was determined by gas chromatography (Figure 6). After the INA pretreatment, no neutral sugars were detected in CDTA and Na2CO3 fractions, probably due to an increment in the uronic acid to neutral sugar proportion in these fractions (Figure 5), which makes the neutral sugar quantification difficult [62]. In these pectin-enriched fractions (Figure 6A,B), the Pph inoculation produced increments in rhamnose (Rha) and arabinose (Ara) concentrations, most likely arising from RG-I. Additionally, Ara and galactose (Gal) also increased in SnCR and TFA fractions in Mock + Pph (Figure 6E,F), pointing also to an enrichment in arabinogalactans.

**Figure 6.** Monosaccharide analysis from different CW fractions: CDTA (**A**), Na2CO3 (**B**), KI (**C**), KII (**D**), SnCR (**E**), and TFA (**F**) of Mock, Mock + Pph, INA, and INA + Pph common bean plants. Monosaccharides are referred as Rha: rhamnose, Fuc: fucose, Ara: arabinose, Xyl: xylose, Man: mannose, Gal: galactose and Glc: glucose. Data represent mean ± SE (*n* = 3). Statistical analysis was carried out by *t*-student for Figure A and B, and by one-way ANOVA for Figure C to F where letters indicate differences by Tukey test (*p* < 0.05). Asterisks refers to non-detected monosaccharides.

In the INA pretreatments a displacement of Rha, Ara, xylose (Xyl), Gal, and glucose (Glc) monosaccharides, from KI, KII, and SnCR fractions to the TFA fraction, was observed (Figure 6C–F), indicating that the polysaccharides implicated in the CW remodeling after priming could be RG, XG, and/or xylans, more tightly linked to the matrix. A decrease in the mannose (Man) content was observed in KI and SnCR fractions after the INA pretreatments compared with Mock. In addition, the Glc increase found in the TFA fraction after the INA pretreatment (Figure 6F) could be related with the higher cellulose content found in crude CWs for those conditions (Figure 4) or even to an increment in other beta-linked-glucans able to reinforce the CW structure such as callose [63,64].

Finally, when plants were inoculated with Pph after priming (INA + Pph), no substantial changes were observed compared with INA, except an increment in Gal and Glc in KI (Figure 6C), and in Ara, Xyl, and Glc in KII fractions (Figure 6D), which could derive from hemicelluloses

### *2.4. INA Priming Increased the Average Molecular Weight of Polysaccharides in All CW Fractions*

Additional information about relative mass distribution and average molecular weights (Mw) of polysaccharides in the CW matrix was obtained by gel permeation chromatography [65] (Figure 7). Mock + Pph involved a Mw increase of all fractions compared to Mock (Figure 7), except in KII, being the most notorious increase in the Na2CO3-pectic fraction (Figure 7B), where polysaccharide dispersion was higher. Interestingly, the INA pretreatment was always associated with a remarkable increase in the estimated Mw of all polysaccharide populations analyzed. This shift was particularly noticeable in hemicellulosic fractions in which a 14-fold (KI fraction) and 6.5-fold increase (KII fractions) was found (Figure 7C,D). In these cases, INA + Pph had nearly negligible impact on Mw compared to INA. These results obtained after INA priming pointed to the possibility of CW remodeling by an extensive polymer cross-linking. Interestingly, the elution profile shown two populations of polysaccharides after the INA pretreatment in fractions CDTA (Figure 7A), KI (Figure 7C) and KII (Figure 7D): (i) relative to lower Mw, which corresponded with Mock and Mock + Pph population, and (ii) of higher Mw, that did not appear in those not treated with INA.

**Figure 7.** Relative mass distribution profiles of CDTA (**A**), Na2CO3 (**B**), KI (**C**), and KII (**D**) fractions extracted from CWs of Mock, Mock + Pph, INA and INA + Pph. 0 and 1 *K*av were assigned to blue dextran and sucrose markers, respectively (indicated with arrows). The average molecular weight in kDa (Mw) obtained by *K*av(1/2) method is shown inserted in boxes.
