Homogalacturonan Pectins Tuned as an Effect of Susceptible rbohD, Col-0-Reactions, and Resistance rbohF-, rbohD/F-Reactions to TuMV

The plant cell wall is an actively reorganized network during plant growth and triggered immunity in response to biotic stress. While the molecular mechanisms managing perception, recognition, and signal transduction in response to pathogens are well studied in the context of damaging intruders, the current understanding of plant cell wall rebuilding and active defense strategies in response to plant virus infections remains poorly characterized. Pectins can act as major elements of the primary cell wall and are dynamic compounds in response to pathogens. Homogalacturonans (HGs), a main component of pectins, have been postulated as defensive molecules in plant–pathogen interactions and linked to resistance responses. This research focused on examining the regulation of selected pectin metabolism components in susceptible (rbohD-, Col-0-TuMV) and resistance (rbohF-, rbohD/F–TuMV) reactions. Regardless of the interaction type, ultrastructural results indicated dynamic cell wall rebuilding. In the susceptible reaction promoted by RbohF, there was upregulation of AtPME3 (pectin methylesterase) but not AtPME17, confirmed by induction of PME3 protein deposition. Moreover, the highest PME activity along with a decrease in cell wall methylesters compared to resistance interactions in rbohD–TuMV were noticed. Consequently, the susceptible reaction of rbohD and Col-0 to TuMV was characterized by a significant domination of low/non-methylesterificated HGs. In contrast, cell wall changes during the resistance response of rbohF and rbohD/F to TuMV were associated with dynamic induction of AtPMEI2, AtPMEI3, AtGAUT1, and AtGAUT7 genes, confirmed by significant induction of PMEI2, PMEI3, and GAUT1 protein deposition. In both resistance reactions, a dynamic decrease in PME activity was documented, which was most intense in rbohD/F–TuMV. This decrease was accompanied by an increase in cell wall methylesters, indicating that the domination of highly methylesterificated HGs was associated with cell wall rebuilding in rbohF and rbohD/F defense responses to TuMV. These findings suggest that selected PME with PMEI enzymes have a diverse impact on the demethylesterification of HGs and metabolism as a result of rboh–TuMV interactions, and are important factors in regulating cell wall changes depending on the type of interaction, especially in resistance responses. Therefore, PMEI2 and PMEI3 could potentially be important signaling resistance factors in the rboh–TuMV pathosystem.


Introduction
The plant cell wall is a highly dynamic and complex structure that undergoes active changes during development, providing support for cell-to-cell communication and responding to biotic and abiotic stresses [1].Plant cell wall heterogenous structures are composed of interacting proteins, polysaccharides, phenolic compounds, and water content, and play a crucial role in determining cell shape and directing growth.Additionally, the plant cell wall serves as a modulating system and signaling hub in plant immunity, particularly during biotic stress conditions [2,3].Extensive research has been conducted on the molecular mechanisms underlying the structural and functional integrity of the cell wall, as well as the perception, recognition, and signal transduction pathways involved in triggered immunity through membrane receptors and effectors [4][5][6][7][8][9].Bellicampi et al. [10] described the cell wall as a battleground where interactions between plant cells and pathogens can lead to either cell wall weakening or reinforcement.Fungi produce a variety of cell wall-degrading enzymes [11], while soft rot bacteria synthesize important wall-degrading enzymes that contribute to their virulence [12].Parasitic nematodes and phytophagous insects also induce wall-degrading enzymes for their invasions [13].Despite the progress in understanding the role and composition of the cell wall in response to damaging intruders, our knowledge of plant cell wall changes and active defense strategies in response to plant virus infections remains incomplete.
Recent research based on transcriptomic and proteomic analyses of plant responses to virus pathogens has shed light on the importance of the cell wall in defense responses to virus stress [14][15][16][17][18][19][20][21][22][23].These studies indicate that genes and transcripts can be significantly differentially regulated during these interactions.Previous research has shown variable distributions of β-1,3-glucanase (PR-2), the catalytic subunit of cellulose synthase A4 (CesaA4), or dynamically changed xyloglucan/xylan metabolism with the participation of xyloglucan transferase XTH-Xet5 in the potato virus y (PVY NTN )-potato pathosystem [24,25].Furthermore, it has been observed that cell wall components' spatial and temporal depositions are strictly associated with the type of reaction to the virus.Studies have also postulated that Solanum tuberosum expansin StEXPa and hydroxyproline-rich glycoprotein (HRGP) extensins are differentially regulated and intracellularly distributed depending on compatible (susceptible) or incompatible (hypersensitive response) interactions between PVY NTN and potato plants [26].These observations confirmed that the susceptible reaction to PVY NTN  was associated with potato cell wall loosening, whereas, during the hypersensitive response, cell wall strengthening was noticed.Additionally, further analyses during the response of NADPH oxidase homologs D and F deficient mutants (rbohD, rbohF, and rbohD/F) to TuMV highlighted cell wall changes [27].The data indicated that not only the symplast but also the apoplast was involved in the activation of the response to potyvirus.According to the observations, the apoplast was dynamically rebuilt during susceptible rbohD-TuMV and Col-0-TuMV interactions, as well as during the limiting virus content in rbohF-TuMV and rbohD/F-TuMV resistance reactions.It was suggested that RbohD could be involved in the resistance response to TuMV, whereas RbohF promotes susceptibility in that pathosystem.Finally, it was reported that glutathione participated in an active reaction to TuMV, confirming the assumption that, in rboh mutants, the apoplast is involved in the signaling defense response to TuMV stress [28].Active changes in the apoplast, and especially alterations in the structure and composition of the cell wall, affected plant resistance to biotic stress [29].
Pectins in the cell wall have many important functions, such as providing the form of the primary cell wall as a polysaccharide matrix component, influencing secondary cell wall structure formation, and supporting cell-to-cell adhesion [30][31][32].Pectins are especially suited for surveillance owing to the activity of pectin methylesterases (PMEs) and other pectin-modifying enzymes [33].Due to their multitude of states and interactions, pectins are presumed to be highly responsive to the environment.In plants, pectins, as major elements of the primary cell wall, are also dynamic compounds in reaction to pathogens.The main pectin components (65%), homogalacturonans (HGs), according to Forand et al. [34], were postulated as defensive molecules in higher plants during infection and have been linked to disease resistance in many reports [2].Moreover, the status of the methylesterification of pectins is a critical factor that directly influences cell wall structure [35].The most important in HG methylesterification/demethylesterification are tuned by two enzymes: PME [EC 3.1.1.11]and proteinaceous PME inhibitors (PMEIs) [36,37], with a diverse effect on pectin biosynthesis.These enzymes primarily participate in stress response, signaling, and defense immunity, including in immune priming reactions [8,38,39].Furthermore, the promotion of the PMEI that counteracts the PME improves disease resistance.According to Liu et al. [40], virus suppression of Gossypium hirsutum GhPMEI3 resulted in a susceptible reaction to Verticillium wilt disease.Other authors suggested that the native PME inhibitor AtPMEI13 can modulate the inhibitory affinity of the PME, which confirmed its defensive potential in plant-insect pathogen infestation [41].Moreover, the screening of mutants with modification of specific cell wall polysaccharides indicated the importance of pectin modulators for penetration of resistance to Colletotrichum [42].Finally, given the numerous functions of pectins and many examples of their differentially regulated modulators involved in susceptibility and/or resistance, it must be emphasized that their role is highly dependent on particular pathogen lifestyles and host plants.Therefore, our studies aimed to examine the regulation of selected Arabidopsis thaliana pectin metabolism components in susceptible rbohD-TuMV and Col-0-TuMV and resistance rbohF-TuMV and rbohD/F-TuMV reactions.This study demonstrated that cell wall rearrangement as an effect of TuMV inoculation affected PME3, PMEI2, and PMEI3, depending on the types of susceptible and resistance responses to TuMV.PME3, but not PME17, was associated with the rbohD-TuMV susceptible reaction leading to low/non-methylesterificated HG distribution, whereas PMEI2 and PMEI3 were important signaling factors in cell wall rebuilding and defense response in the rbohF-TuMV and rbohD/F-TuMV pathosystem.

Results
2.1.Virus Content and Significant Ultrastructural Apoplast Changes in TuMV Infected Col-0, rbohD, rbohF, and rbohD/F The ultrastructural analyses of the apoplast area between 3 and 21 days post-TuMV inoculation indicated significant changes compared to susceptible (rbohD, Col-0) (Figure 1A-C,E-G) and resistance (rbohF, rbohD/F) reactions (Figure 1I-K,M-O), as well as infected and mock-inoculated plants (Figure 1D,H,L,P).The Col-0 and mutant plants exhibited different levels of TuMV (Figure S1).Virus titers, based on the expression of the TuMV-CP gene, steadily increased in Col-0 and rbohD plants, whereas in rbohF and rbohD/F plants, they dynamically decreased between 3 and 21 dpi (Figure S1).In susceptible interactions, the formation of paramural bodies was observed starting from 3 dpi (Figure 1A,E).Moreover, multivesicular structures were formed from the cell wall to the vacuole between 7 and 21 dpi of virus inoculation (Figure 1B,C,F,G), accompanied by changes in cell wall structure and thickening not only around the plasmodesmata area.Dynamic formation of paramural bodies during the resistance reaction was observed between 3 and 21 dpi, especially in the epidermis (Figure 1I,M).Seven days post-TuMV inoculation, cell wall structures were rebuilt in the mesophyll as well as vascular tissues (Figure 1J,N).Furthermore, thicker cell walls than those in susceptible and mock-inoculated plants were noticed in mesophyll and vascular tissues at the 21 dpi timepoint (Figure 1K,O).

Crucial Modulation of Relative Expression of
Selected AtPME, AtPMEI, and AtGAUT1, AtGAUT7 Genes in Resistant (rbohF and rbohD/F) and Susceptible (rbohD, Col-0) TuMV-Infected Plants Ultrastructural analyses of Col-0 and rboh-mutant plants infected with TuMV focused on cell wall changes, which were in line with our previous investigation on glutathione content in the apoplast [28] during different reactions to the virus.To examine the potential association of genetic factors in cell wall remodeling, we selected pectin metabolismassociated genes, such as pectin methylesterase 3 and 17 (AtPME3 and AtPME17), pectin methylesterase inhibitors 2 and 3 (AtPMEI2 and AtPMEI3), and galacturonosyltransferases 1 and 7 (AtGAUT1 and AtGAUT7), for analysis of relative expression.In the current literature, pectin methylesterases (AtPME3, AtPME17), and methylesterase inhibitors (AtPMEI2 and AtPMEI3) were reported as key elements for susceptibility or resistance in different pathogen interactions [43][44][45][46][47].Moreover, we also chose the AtGAUT1 and AtGAUT7 genes, considering that the proteins encoded by these genes are critical for HG pectin synthesis [48].Analyses of normalized relative gene expression for all selected Arabidopsis thaliana genes at 3, 7, and 21 dpi were performed and indicated an interesting connection between the type of plant reaction to TuMV.AtPME3 and AtPME17 relative expression (Figure 2A Moreover, the highest level of expression for both analyzed AtPMEIs was noticed in rbohD/F-TuMV.In contrast, in Col-0-TuMV and rbohD-TuMV, a significant decrease in AtPMEI2 and AtPMEI3 between 3 and 21 dpi was reported.Moreover, the highest reduction was observed for rbohD-TuMV plants (about 20.64 fold and 18.35 fold, respectively, for AtPMEI2 and AtPMEI3).These results indicated that the upregulation of AtPMEI2 and AtPMEI3 was associated with an increased resistance reaction typical for rbohF and rbohD/F against TuMV.A similar connection with the resistance reaction was also shown by expression analyses of AtGAUT1 and AtGAUT7 (Figure 2E,F).Both of these genes were highly upregulated in rbohF-TuMV and rbohD/F-TuMV interactions between 3 and 21 dpi (Figure 2E,F).Moreover, the highest induction of 1.74 fold (for AtGSTU1) and 1.39 fold (in the case of AtGSTU7) was observed in rbohD/F-TuMV in contrast to susceptible Arabidopsis plants (Col-0 and rbohD).The AtGAUT1 gene expression was significantly downregulated between 3 to 21 dpi in the susceptible reaction (Figure 2F).The lowest expression levels were revealed at 21 dpi in rbohD-TuMV, and downregulation was about 7.59 fold between 3 to 21 dpi, whereas the expression of AtGAUT7 did not significantly change in Col-0-TuMV and rbohD-TuMV plants compared to mock-inoculated plants.These findings indicated that the resistance reaction against TuMV required upregulation of two AtPMEIs (AtPMEI2 and AtPMEI3) and two AtGAUTs (AtGAUT1 and AtGAUT7) in rbohF and rbohD/F mutants.Furthermore, the resistance reaction to TuMV was also associated with no significant change in AtPME17 expression and downregulation of AtPME3.This fact may confirm that the synthesis and presence of methylesterificated homogalacturonan pectins can be important in resistant plants with control of the demethylesterification process catalyzed by AtPME3.Taking into account results obtained from the relative expression of selected genes associated with pectins in different reactions to TuMV, PME3, PMEI2, PMEI3, and GAUT1 proteins were localized at the ultrastructural level by quantified immunogold labeling.Considering the changes in gene expression tendency, we selected for this step 7 and 21  Taking into account results obtained from the relative expression of selected genes associated with pectins in different reactions to TuMV, PME3, PMEI2, PMEI3, and GAUT1 proteins were localized at the ultrastructural level by quantified immunogold labeling.Considering the changes in gene expression tendency, we selected for this step 7 and 21 days past virus inoculation.Moreover, based on data presented by Atmodjo et al. [48], GAUT1 can act in plant cells with GAUT7, but our data showed that the level of AtGAUT1 expression was more highly induced than AtGAUT7 in resistant plants.Therefore, we decided to select GAUT1 for localization.Firstly, the potential subcellular localization was checked by using the bioinformatic server SUBA5, which connects most protein predictor localization systems and new data from mass spectrometry assay (MS/MS) to predict the potential localization of A. thaliana proteins.SUBA program consensus (SUBAcon) indicates potential protein localization.For PME3, PMEI2, and PMEI3, SUBAcon suggests extracellular/cell wall localization, whereas for the GAUT1 protein, the Golgi network has been indicated (Table S3).However, bioinformatic predictors connected with SUBA5 adding MS/MS data indicated that the localization of those proteins in cells can be wider than suggested only by SUBAcon (Table S3).Therefore, we performed direct immunogold localization of these proteins.
The immunogold localization of PME3 revealed statistically significant differences between susceptible (Figure 3A,B,E,F,M) and resistant rboh plants' reactions (Figure 3C,D,G,H,M).Moreover, in general, virus inoculation changed the PME3 deposition in Arabidopsis leaf tissues compared to the control (Figure 3I-M).However, the localization of PME3 between 7 to 21 dpi was significantly upregulated in TuMV-inoculated Col-0 (1.35 fold) and Col-0-rbohD (1.16 fold).These results are in line with relative AtPME3 gene expression, where the most intense induction of AtPME3 was observed in rbohD-TuMV and Col-0-TuMV at 7 and 21 dpi (Figure 3A,B,E,F,M).Furthermore, in the susceptible reaction, PME3 was localized not only in the cell wall, as in mock-inoculated Arabidopsis leaf tissues (Figure 3I,J), but also in the cytoplasm near the virus cytoplasmic inclusion (Figure 3A,E,F).In rbohD, where the localization was the most intense, PME3 deposition in the rebuilt cell wall was accompanied by the location in multivesicular structures, paramural bodies, and chloroplasts with a vacuole (Figure 3B,F).In comparison, the rbohF and rbohD/F plants after virus inoculation showed a decrease in PME3 between 7 and 21 dpi, respectively, of 1.33 fold and 1.7 fold (Figure 3M).In resistant rbohF and rbohD/F, PME3 deposition was weak, and its location was noticed in paramural bodies near the rebuilt cell wall and cytoplasm (Figure 3C,D,G,H).Moreover, the weakest localization, even lower than in mock-inoculated rbohF and rbohD/F plants (Figure 3K,L), was noticed in rbohD/F-TuMV inoculation (Figure 3M).
On the other hand, downregulation of PMEI2 and PMEI3 deposition between 7 and 21 dpi (Figure 4A-D,F-I,U,V) was revealed in Col-0 and rbohD plants.PMEI2 and PMEI3 were localized in the cell wall with induced paramural bodies, as well as in the cytoplasm and vacuole (Figure 4A,B,F,G).However, at the 21 dpi timepoint, they were mainly located around the cell wall area in the mesophyll or vascular tissue (Figure 4C,D,H,I).Moreover, the highest reduction was characterized for rbohD plants with the virus at 21 dpi (about 1.23 fold and 1.37 fold, respectively, for PMEI2 and PMEI3) (Figure 4F-I,U,V), and the localization at this timepoint was sometimes even lower compared to mockinoculated rbohD or Col-0 (Figure 4C-E,H-J).In contrast, PMEI2 and PMEI3 were both upregulated in virus-inoculated rbohF (2.08 fold and 1.15 fold, respectively, for PMEI2 and PMEI3) and rbohD/F (1.29 and 1.17 fold, respectively) between 7 and 21 dpi (Figure 4U,V).Moreover, both inhibitors' epitopes were deposited in the cell walls with paramural bodies (also with plasmodesmata) and decorated multivesicular and membranous structures in the cytoplasm, as well as ER, Golgi network, and vacuoles in all leaf tissues (Figure 4K-N,P-S).Furthermore, the localization was more intense at 21 dpi than at 7 dpi after TuMV inoculation, as well as compared with mock-inoculated tissues (Figure 4K-T).ies (also with plasmodesmata) and decorated multivesicular and membranous structures in the cytoplasm, as well as ER, Golgi network, and vacuoles in all leaf tissues (Figure 4K-N,P-S).Furthermore, the localization was more intense at 21 dpi than at 7 dpi after TuMV inoculation, as well as compared with mock-inoculated tissues (Figure 4K-T).The most intense localization level of epitopes of PMEI2 and PMEI3 was observed in rbohD/F plants with TuMV (Figure 4P-S,U,V).These results strictly correspond with the relative gene expression, suggesting that the upregulation of the genes/proteins AtPMEI2/PMEI2 and AtPMEI3/PMEI3 was associated with increased resistance and the resistance reaction of rbohF and rbohD/F against TuMV.
To evaluate A. thaliana GAUT1, which is responsible for HGs synthesis, immunogold labeling localization during TuMV infection at 7 and 21 dpi timepoints, when the AtGAUT1 relative gene expression was the most induced, was conducted.In mock-inoculated Col-0 and rboh-mutant plants, GAUT1 was observed in the plasma membrane, Golgi network, and vacuole (Figure 5).The GAUT1 protein deposition was induced in virus-inoculated rbohF and rbohD/F plants between 7 and 21 dpi compared to mock-inoculated plants, as well as to susceptible Col-0 and rbohD (Figure 5A-M).The highest induction (1.25 fold) was observed in TuMV-rbohD/F (Figure 5D,H,M).Moreover, the GAUT1 in resistant TuMV-rbohF and TuMV-rbohD/F was mainly localized in the trans-Golgi network and plasmalemma with vesicular/membranous structures (Figure 5C,D,G,H).On the other hand, the plants with susceptibility, Col-0 and rbohD, reacted with downregulation of GAUT1 epitope localization (Figure 5A,B,E,F,M).Moreover, the lowest localization of GAUT1 was reported at 21 dpi in TuMV-rbohD plants, and the reduction was about 1.54 fold between 7 and 21 dpi.The GAUT1 location documented in TuMV-Col-0 and TuMV-rbohD between 7 and 21 dpi was even less intense than in mock-inoculated Col-0 and rbohD plants.Furthermore, GAUT1 in the susceptible reaction was deposited along with the plasmalemma, in the cytoplasm near the cell wall and plasmodesmata (Figure 5A,B,E,F).The obtained results suggested that the induction of relative AtGAUT1 and AtGAUT7 gene expression corresponded with Arabidopsis GAUT1 protein deposition and was related to both the resistance interactions: rbohF-TuMV and especially rbohD/F-TuMV.

Highly Methylesterificated HGs' Domination in Resistance Reactions to TuMV
In the analysis of PME activity and its correlation with cell wall methylester levels during susceptible Col-0-TuMV, rbohD-TuMV, and resistance rbohF-TuMV, rbohD/F-TuMV indicated higher demethylesterification activity in susceptible Col-0-TuMV, and especially rbohD-TuMV interactions.The highest level of PME activity was detected at 21 days post-inoculation with TuMV in both susceptible interactions.We checked the tendency in localization between high and low/non-methylesterificated HGs expressed through JIM7 and JIM5 detection during both types of reaction to TuMV.The immunogold localization (Figure 6A-L) confirmed by quantification (Figure 6M) indicated that low/non-methylesterificated HGs were deposited more intensely during susceptible TuMV interactions with rbohD mutant and Col-0 plants compared to both resistance types of interaction (rbohF, rbohD/F) as well as mock-inoculated plants (Figure 6E-J).These low/nonmethylesterificated HG pectins were found in the changing cell wall, near the plasmalemma and plasmodesmata in rbohD and Col-0 plants infected with TuMV.In contrast, in resistance interactions with rbohF and rbohD/F mutants, the most intense deposition was observed for highly methylesterificated HGs compared to mock-inoculated plants (Figure 6A-D,K,L).These highly methylesterificated HGs were located near the plasmalemma with paramural bodies and in multivesicular structures (Figure 6C,D,G,H).These findings suggest that demethylesterification of HG pectins is closely associated with susceptible interac-tions, especially in rbohD mutants and Col-0 plants, while intense accumulation of highly methylesterificated HG pectins is characteristic of resistance interactions with rbohD/F and rbohF mutant plants.

Induction of PME Activity in Susceptible Interaction with a Decrease of Cell Wall Methylesters Level
Validation of PME activity revealed a significant increase between 7 and 21 dpi in virus-inoculated susceptible (Col-0) and increased susceptibility plants (rbohD), partially corresponding to increased expression of AtPME3.The activity increased by 1.31 fold and 1.37 fold for Col-0 and rbohD plants with the virus between 7 and 21 dpi (Figure 7).In contrast, PME activity was downregulated in virus-inoculated rbohF and rbohD/F plants between 7 and 21 dpi, with the lowest activity observed in rbohD/F plants with the virus at 21 dpi (Figure 7).This trend corresponded with the estimation of cell wall methylester levels (Figure 8), which increased in plants showing an increased resistance reaction (rbohF, rbohD/F) to TuMV between 7 and 21 dpi, with the highest level in rbohD/F-TuMV plants (1.19 fold) (Figure 8).In contrast, Col-0 and rbohD-TuMV plants exhibited downregulation of methylester levels, with the highest reduction occurring in rbohD-TuMV (1.47 fold) between 7 and 21 dpi (Figure 8).These results suggest that the demethylesterification process is associated with the susceptible reaction of rbohD and Col-0 plants to TuMV.

Induction of PME Activity in Susceptible Interaction with a Decrease of Cell Wall Methylesters Level
Validation of PME activity revealed a significant increase between 7 and 21 dp virus-inoculated susceptible (Col-0) and increased susceptibility plants (rbohD), part corresponding to increased expression of AtPME3.The activity increased by 1.31 fold 1.37 fold for Col-0 and rbohD plants with the virus between 7 and 21 dpi (Figure 7 contrast, PME activity was downregulated in virus-inoculated rbohF and rbohD/F pl between 7 and 21 dpi, with the lowest activity observed in rbohD/F plants with the v at 21 dpi (Figure 7).This trend corresponded with the estimation of cell wall methyle levels (Figure 8), which increased in plants showing an increased resistance reac (rbohF, rbohD/F) to TuMV between 7 and 21 dpi, with the highest level in rbohD/F-Tu plants (1.19 fold) (Figure 8).In contrast, Col-0 and rbohD-TuMV plants exhibited do regulation of methylester levels, with the highest reduction occurring in rbohD-Tu (1.47 fold) between 7 and 21 dpi (Figure 8).These results suggest that the demethyles fication process is associated with the susceptible reaction of rbohD and Col-0 plan TuMV.

Discussion
NADPH oxidases in plants can regulate or modulate responses to various plantpathogen interactions [49].Previous findings have shown varied reactions of rbohD, rbohF, and rbohD/F transposon mutants to TuMV infection [27].Systemic TuMV infection was promoted in rbohD mutants, indicating that RbohD is important in TuMV infection.Moreover, virus concentration was significantly induced in rbohD mutants more dynamically than in Col-0 plants (Figure S1), and it was also shown by [27].Furthermore, rbohD-TuMV plants displayed reduced ROS, decreased glutathione content, and higher levels of PR-1 proteins, which can act as markers of TuMV infection in this pathosystem [27,28].Conversely, rbohF and rbohD/F mutants exhibited a significant reduction in TuMV infection accompanied by H 2 O 2 induction compared to mock-inoculated plants.These results are compatible with a significantly dynamic increase in total glutathione content in cells and the apoplast, along with upregulation of GGT enzyme activity [28].This observation suggests that RbohF is important in TuMV infection or may even increase susceptibility to this virus.Ultrastructural analyses of rbohD, rbohF, and rbohD/F interactions with TuMV have focused on changes in the plant cell wall.Previous research has mainly concentrated on cell wall changes in plant-pathogen interactions involving bacteria and fungi as mechanical and enzymatic destructors of the host apoplast [50][51][52].However, proteins and enzymes involved in active cell wall defense responses during plant virus infection may play an important function [14,18].Research on potato plants with different levels of resistance to PVY NTN has shown that virus infection induces active cell wall rearrangements during compatible and incompatible interactions [24].It was revealed that β-1,3-glucanase, cellulose synthase catalytic subunit CesaA4, and hydroxyproline-rich glycoproteins A4 (StEXTA4-HRGP) with potato expansin A3 (StEXPA3) are actively modulated as an effect of PVY NTN inoculation [24,26].Given these findings and the observed cell wall rearrangements during the reaction of rbohD and rbohF mutants to TuMV, we decided to explore in more detail selected components of pectin metabolism in the rboh-TuMV pathosystem.
Ultrastructural analysis of susceptible rbohD and Col-0 interactions with TuMV documented the active formation of paramural bodies in mesophyll and vascular tissues during systemic infection.Additionally, a more expanded cell wall structure was observed, especially in the plasmodesmata area, accompanied by the formation of multivesicular structures and the presence of virus particles and virus cytoplasmic inclusions.These observations are consistent with other pathosystems where cell wall alterations lead to the formation of paramural bodies attached to the plasma membrane [53,54].Cytological modifications also include the formation of vesicles and tubules originating from the border cell wall and incorporating into the plasmalemma, as postulated in potato virus Y NTN (PVY NTN ) and potato virus M (PVM) infections [24,55,56].In contrast, the resistance reactions of rbohF and rbohD/F mutants to TuMV displayed significant cell wall rebuilding.Paramural bodies were forming, especially in the epidermis.The cell wall in the resistance reaction was visibly thicker in mesophyll and phloem cells, accompanied by the deposition of phenolic-like compounds in xylem tracheary elements.These ultrastructural observations are in accordance with alterations induced by Botrytis cinerea, Colletotrichum, or Blumeria, where phenolic substances accumulate around cell walls and may act as a barrier for pathogens [53,57,58].Moreover, similar cell wall thickening in resistance reactions was documented during PVY NTN and PVM-potato interactions [24,26,55].Furthermore, according to data presented by O'Brien et al. [59] and Tse et al. [60], the strengthening of the plant cell wall plays a key role in plant defense and could be accompanied by the induction of wall material-associated vesicles, such as paramural bodies or multivesicular structures.Conversely, as postulated by Tse et al. [60], these structures can be arranged in degradation in the vacuole or are fused to the plasmalemma to release vesicles.Plant cell wall remodeling is a dynamic process in different interactions as well as in normal growth and development [61].It is a well-known fact that the plant cell wall is the first barrier against pathogens, and its integrity plays an important role in plant defense [62].Moreover, the remodeling of cell wall components is a response to exposure to biotic stress [63].Transcriptomic profiling and microarray analyses of gene expression in susceptible and resistance responses to some groups of viruses have reported that cell wall-related genes and transcripts are regulated during plant-virus infection [15][16][17]64].
Pectins are major components of the primary cell walls of dicotyledonous and nongramineous monocotyledonous plants, accounting for about 35% of total cell wall polysaccharides [65].Pectins also act as active molecules in many cellular metabolism pathways, physiological processes, and signaling processes [66,67].There are mainly four different types of pectin polysaccharides; the major ones are HGs, rhamnogalacturonans I (RGsI), the most structurally diverse rhamnogalacturonans-II (RGsII), and covalently linked RG-I xylogalacturonans (XGas) [68].However, HGs are the most abundant pectins, constituting 65%, and recent findings underline the importance of pectins as defensive components in plants during pathogen infection [21,37,69].HGs are synthesized in the Golgi network from nucleotide sugars and are secreted in a methylesterificated form into the cell wall [70].Their structure can be modified by homogalacturonan-modifying enzymes (HGMEs) [71], such as pectin methylesterases (PME) [E.C. 3.1.1.11].In Arabidopsis, PMEs belong to multigenic (66 A. thaliana genes) and super-multiple enzyme families [72].PMEs selectively catalyze the process of demethylesterification in HGs and can lead to stiffness or softness of the cell wall-pectin matrix [38,73].However, as postulated by Coculo et al. [74], the generation of low degrees of methylesterificated HG pectins may promote proton-releasing and depolymerization conducted by enzymes such as polygalacturonases (PGs) and pectate lyase-like proteins (PLLs), resulting in cell wall loosening and expansion.Namely, it was documented first by Juge et al. [75] that PME activity is modulated by a family of proteinaceous inhibitors known as PMEIs, which belong to multigenic families in Arabidopsis (77 putative PMEI genes).Therefore, it can be assumed in line with Hocq et al. [73] that PMEs with PMEI enzymes exhibit diverse effects on physiology and pectin biosynthesis.Moreover, the specific PME-PMEI relation is a critical factor in the fine-tuned degree of demethylesterificated HG pectins and that aspect may determine cell wall remodeling and integrity not only during the biological process, but also in plant-pathogen interactions [39].Based on those statements and taking into account that other genes/enzymes related to pectin metabolism (such as PG), PLL, as well as pectin acetylesterase PAE [E.C. 3.1.1.6]were shown to be rather not induced by plant viruses or endosymbionts [70].Therefore, we decided to examine normalized relative gene expression of selected PME and PMEI during rboh-TuMV interactions.Our findings indicated that AtPME3 was highly induced in the susceptible Col-0-TuMV interaction.However, in the rbohD-TuMV interaction, the upregulation of AtPME3 was the most intense.Conversely, regardless of the interaction type, AtPME17 gene expression was unchanged compared to mock-inoculated plants.AtPME17 has been implicated in interactions in different pathosystems [44,74].Interestingly, Arabidopsis knockout mutant pme17 modified the trophic behavior of Myzus persica [70].However, PME17 expression is significantly increased with other aphids and whiteflies [76,77].Furthermore, it was postulated that PME17 could participate in facilitating the progression of the stylet in wild-type plants.It seems that AtPME17 is not engaged in the response to TuMV, whereas AtPME3 may be responsible for pectin demethylesterification during susceptible interactions with TuMV in rbohD and Col-0 plants.
Consequently, the highest induced PME activity was revealed in rbohD-TuMV and Col-0-TuMV compared to mock-inoculated and resistant rbohF-TuMV and rbohD/F-TuMV plants.In Arabidopsis, some PME isoforms show altered expression in response to Botrytis cinerea [78].Specifically, AtPME3 is induced during infection with B. cinerea and P. carotonorum, acting as a susceptibility factor [43].Furthermore, the expression of PME genes and PME activity in plant-virus interactions has broader implications.For example, the StPME gene in potato Igor was upregulated as a result of PVY NTN inoculation, but it was estimated only at 0.5 h post-inoculation [79].It is in line with our observation, where the susceptible reaction to TuMV induced AtPME3 gene expression.Conversely, AtPME3 was downregulated during interaction with CaLCuV at 12 dpi [80].Additionally, Yang et al. [81] indicated that the AtPME3 gene in TuMV-Arabidopsis was downregulated in tissues from the inoculation point (zone 0), while induction of AtPME3 was observed millimeters away from the inoculation point, in zone 3.In tobamovirus interactions, PME proteins actively participate in TMV local transport through a requirement for virus plasmodesmata-exploring movement [82].Specifically, the binding of TMV MP may interfere with PME activity, modifying the cell wall ion balance, inducing changes in plasmodesmata permeability, and facilitating cell-to-cell movement [83].Moreover, Chen and Citovsky [84] have indicated that PME is also involved in TMV systemic movement, aiding in the virus's exit from the phloem to adjacent tissues of noninfected parts of plants.
Recent reports have highlighted the stimulation of expression of multiple methanolinducible genes (MIGs), which affects the permeability of plasmodesmata and is a consequence of pectin demethylesterification [85][86][87].It has also been highlighted by Andika et al. [88] that PME interacts with turnip vein clearing virus (TVCV), cauliflower mosaic virus (CaMV), and even Chinese wheat mosaic virus (CWMV) at the cell wall, and the results of these interactions can be essential for virus cell-to-cell movement.PME silencing or overexpression of PMEI in Nicotiana has been shown to delay TMV and TVCV systemic movements and significantly reduce host susceptibility [89,90].Similar to our observations in the rbohD-TuMV and Col-0-TuMV interactions, Dorokhov et al. [91] reported that a cell wall enriched in pectin can be more flexible and may allow dynamic changes in plasmodesmata structure [92][93][94].Moreover, PME is involved in HG demethylesterification in muro accumulated in the cell wall near plasmodesmata [87,95].Consequently, pectin demethylesterification expressed through AtPME3 upregulation and PME activity induction, with PME3 deposition on an ultrastructural level like in rbohD-TuMV, seems to influence cell wall expansion changes in the plasmodesmata area, leading to virus transport from cell to cell, including to vascular tissues.In susceptible interactions with rbohD and Col-0-TuMV, a natural consequence of induced AtPME3 gene expression and PME activity was the intense and statistically significant deposition of PME3 in rebuilt cell walls and multivesicular bodies compared to mock-inoculated Arabidopsis leaf tissue, and compared to resistance reactions rbohF-TuMV and rbohD/F-TuMV, has been observed.Interestingly, potyvirus cytoplasmic inclusion (CI) proteins can act as structures docking movement complexes to plasmodesmata [96,97].Moreover, CI and the viral protein P3N-PIPO complex coordinate the formation of PD-associated structures that facilitate intercellular movement.The localization of PME3 documented during TuMV stress in the plasma membrane and vesicular structures is partly similar to observations presented by Morvan et al. [95] based on Linum PME in cortical tissue.Derksen et al. [98] and Morvan et al. [95] reported PME epitopes distributed along the plasmalemma and used antibodies that recognized epitopes of PME precursors in the Golgi network.The authors underlined that PME can be retained at the plasma membrane and also secreted into vesicular structures.
The detection of low/non-methylesterificated and highly methylesterificated HG fractions of pectins indicated that in the susceptible reactions of rbohD and Col-0 plants to TuMV, there was statistically significant domination of low/non-methylesterificated HGs.This was confirmed by the quantification of immunogold labeling.These low/nonmethylesterificated HGs associated with the susceptible reaction to TuMV were located in the changed cell wall, as well as in the plasmalemma and near plasmodesmata.Moreover, the observation indicated that PME3 labeling in the rbohDand Col-0-TuMV interactions are correlated with those obtained with low/non-methylesterificated HGs (JIM5), although the quantification indicated much more intense deposition of PME3.Generally, different authors have postulated that the deposition of HGs (detected by JIM5 and JIM7) is consistent with a weak signal [99,100].According to Libermann et al. [101], low methylesterificated pectins through JIM5 detection in mung bean hypocotyl were documented in the Golgi network and vesicular structures.Our findings regarding the susceptible rbohD-TuMV and Col-0-TuMV interaction are in line with statements presented by Lionetti et al. [47] that the demethylesterification process of HGs by PME activity might result in a decrease in cell wall strength, acting as a barrier that favors virus pathogen invasion.Quite interesting observations were documented by Fan et al. [102], who found that a high abundance of demethylesterificated HGs and highly demethylesterificated pectins were correlated with low pathogenicity in bananas during infection with Fusarium oxysporum f. sp.cubense pathogenic races 1 (Foc1) and 4 (Foc4) on banana (Musa AAA, Brazilian).Furthermore, during interactions between host and pathogen, some authors have reported a decrease in HGs recognized by JIM5 and JIM7, and this process is involved in the response of most plants to pathogens [100,102,103].However, opposite relationships have also been presented [104,105].Therefore, it can be assumed that plants finely tune pectin methylesterification levels and regulate PME activity during their development.In plant-pathogen interactions, the cooperation between PME and PMEI with the degree of HGs methylesterification is associated with plant resistance to pathogens [45,106].
In general, the degree of methylesterification of HGs can play a role in determining the biomechanical properties of the cell wall [107,108].Demethylesterification can result in forming calcium bands with other HG molecules, leading to an egg-box structure that underlies the core of the pectin gel formation process [109].Calcium-linked HG increases cell wall hydration [110].Moreover, Tibbits et al. [111] postulated that the strength of pectin gels is highly related to the free calcium ions in the apoplast, and the stiffness of this kind of gel can be reduced through the disassociation of calcium crosslinks.Conversely, partially demethylesterificated HGs can be easily targeted by pectin-degrading enzymes (like PG or PLL), which is especially important in bacteria and fungi interaction [72].Otherwise, PMEI coordinated with PME is a crucial factor in regulating effects on cell wall structure properties, such as strengthening, loosening, and modulation response to virus infection, especially immune response.Our findings indicated that resistance reactions with reduction of virus content and cell wall rearranged structure in rbohF and rbohD/F to TuMV were associated with significant induction of two examined Arabidopsis PMEI genes: AtPMEI2 and AtPMEI3 (between 3 and 21 dpi).Moreover, the normalized relative gene expression of AtPMEI2 and AtPMEI3 was confirmed by dynamically increased PMEI2 and PMEI3 protein deposition at the ultrastructural level.Furthermore, the highest induction of both gene expression and protein deposition was reported in the rbohD/F-TuMV reaction.That tendency in rbohD/F-TuMV and rbohF-TuMV interaction was also accompanied by an intense decrease in PME activity.These results seem to be partly in line with a completely different pathosystem, Arabidopsis thaliana with Botrytis cinerea, presented by Lionetti et al. [45], where expression of AtPMEI1 and AtPMEI2 downregulated PME activity.However, in contrast to our observation, reduced PME activity resulted in an increased level of demethylesterification of HGs.It should be highlighted that pathogen secretion of cell-wall-degrading enzymes, which hydrolyze pectins, is an important step to successful plant infection by bacteria or fungi, but not for viruses [43,112,113].Factually, plantinduced PME activity and lower levels of pectin methylesterification were observed in these interactions correlated with reduced demethylesterification of pectins like necrotrophic pathogens and/or Pseudomonas syringae [43,105].Moreover, Lionetti et al. [47] postulated that upregulation of AtPMEI10, AtPMEI11, and AtPMEI12 were characterized for response to B. cinerea infection, whereas pmei10, pmei11, and pmei12 mutants displayed an increase in PME activity and decreased demethylesterification of pectins, which led to increased lesion formation during Botrytis infection.The tendency observed by Lionetti et al. [47] and Coculo and Lionetti [38] indicated that plants modulated PME activity by the expression of PMEI in response to infection.Moreover, it was pointed out that AtPMEI10, AtPMEI11, and AtPMEI12 can act as mediators of cell wall-induced plant immunity [38].Furthermore, it was documented that PMEI can play a role as an antimicrobial factor/protein against Fusarium, Alternaria brassicicola, Xanthomonas campestris, or Pseudomonas syringae [114].On the other hand, overexpression of PMEI1 and PMEI2 in Arabidopsis displayed resistance to powdery mildew and soft rot disease [45].It has been shown that overexpression and high induction of PMEI enhanced plant resistance in different pathosystems [40,105].According to presented statements, overexpression of PMEIs in Nicotiana as well as in Arabidopsis counteracts PMEs, leading to increased resistance to tobacco mosaic virus and turnip veinclearing virus infection [46].From these statements and obtained results in rbohF-TuMV and rbohD/F-TuMV pathosystems, it can be concluded that an increase of PMEI can influence reducing virus transport through downregulation of PME activity and even by hindering the enlargement of plasmodesmata.
In addition to PME-PMEI regulation, rbohF-TuMV and rbohD/F-TuMV interaction was characterized by upregulation of relative gene expression AtGAUT1 and AtGAUT7 accompanied by induction of highly methylesterificated HGs.Finally, an increase in cell wall methylesters level was revealed in both rbohF-TuMV and rbohD/F-TuMV interaction.It is indicated that induction of GAUT1 and GAUT7 gene expression, as seen in methylesterificated HGs synthesis [48], is in line with deposition of HG levels in both interactions, in contrast to susceptible rbohD-TuMV and Col-0-TuMV.Moreover, it was also reported that there is a close correlation between areas of deposition of GAUT1 and highly methylesterificated HGs (through JIM7) on the ultrastructural level during both resistance reactions to TuMV.However, it was postulated by Li et al. [115] that highly methylesterificated HGs were deposited at a 6 h timepoint during the interaction between resistance bananas on Foc4 (Fusarium oxysporum f., sp.cubense).On the other hand, Simon et al. [104] reported highly methylesterificated HGs recognized by JIM7 were induced during leaf pathogen Cymadathea trifollii infection.Furthermore, the significantly most intense localization of highly methylesterificated HGs was noticed in rbohD/F-TuMV leaf tissues accompanied by GAUT1, and was observed in the Golgi network, plasma membrane, and vesicular, membranous structures closely around and in the cell wall.However, the deposition of highly methylesterificated HGs together with GAUT1 indicated active distributions of HGs in resistance reactions to TuMV.HG is usually synthesized in the Golgi network [72,116] and can then be secreted into the cell wall in a highly methylesterificated form [117]. Engagement of plasma membrane and vesicular structures in GAUT and highly methylesterificated HG localization may confirm active distribution.Therefore, it can be assumed that during active cell wall rebuilding, AtPMEI2 and AtPMEI3 participated in rbohF and especially rbohD/F resistance reactions to TuMV, and it can be the effect of PME reduction activity.Moreover, reduced PME activity in that reaction resulted in a high content of cell wall methylesters and highly methylesterificated HGs.Induction of highly methylesterificated HGs seems to contribute to resistance reactions to TuMV-it modulates reduction in virus content or even virus transport.
Generally, many PMEI members can suggest the presence of dedicated, direct pairs of the specific PMEI-PME interactors, which can be an important factor in modulating demethylesterification in HG pectins [118] that determine cell wall rebuilding.Additionally, this fact can indicate that different PMEIs, like PMEI2 and PMEI3 in rboh-TuMV interactions, can target different PMEs to induce TuMV-dependent cell wall changes.This is a promising finding that needs further biochemical confirmation of a direct interaction between PMEI2 and PMEI3 with/or without PME3 as a response to TuMV.

Plant Material, Virus Inoculation, and Molecular Verification of TuMV Content
Cell wall remodeling in pectin-associated elements was checked in A. thaliana (L.) Heynh wild-type (Col-0) plants and selected specific mutants: A. thaliana rbohD, rbohF, and rbohD/F [27,28,119].The Col-0 and mutant plants were selected based on different reactions to TuMV infection presented previously by Otulak-Kozieł et al. [27] and Otulak-Kozieł et al. [28].All homozygous mutant seeds were kindly provided by Miguel-Angel Torres Laboratory.The plants were sown and cultivated according to the procedure and conditions presented by Otulak-Kozieł et al. [27,28].All plants were next mock or TuMV inoculated, as described by Otulak-Kozieł et al. [120], Tomilson [121], and Walsh and Jenner [122].For virus inoculation, we used TuMV inoculum (isolate PV-0104 was kindly provided by Leibniz Institute, Braunschweig, Germany) in phosphate buffer, as was presented [28,123].The Col-0 and mutant plant leaves after 3, 7, and 21 dpi (days post-inoculation) of mockinoculation and TuMV-inoculation were checked for the presence of the virus using qPCR (quantitative polymerase chain reaction).The verification of TuMV was based on the expression of the TuMV-CP gene in comparison to the mean expression of the plant host reference genes, AtEf1α and AtF-Box, as presented by Otulak-Kozieł et al. [28] and Otulak-Kozieł et al. [120], with use primers presented by Arous et al. [124] for TuMV-CP.The number of plants selected for all analyses was the same as presented by Otulak-Kozieł et al. [28] and performed in triplicate using a new set of plants every time.The mockinoculated plants were free of TuMV.The results of the verification of the TuMV content are in Figure S1.

Analysis of Relative Expression of Selected AtPME, AtPMEI, and AtGSTU Genes in
TuMV-Infected Col-0, rbohD, rbohF, and rbohD/F Plants Using qPCR During the investigation of changes in pectin-associated elements associated with cell wall modification during TuMV infection, we performed the validation of the expression of selected genes.For analyses, we selected Arabidopsis genes: AtPME3, AtPME17, AtPMEI2, AtPMEI3, AtGAUT1, and AtGAUT7.The selection of genes for analyses was made based on two criteria: direct involvement of these genes in plant-pathogen interactions or a crucial role in pectin formation during cell wall building.Direct involvement in plant pathogen was reported in AtPME3, AtPME17, AtPMEI2, and AtPMEI3, respectively, by Raiola et al. [43], Del Corpo et al. [44], Lionetti et al. [45], Lionetti et al. [46], and Lionetti et al. [47].For the core role in the synthesis of pectin homogalacturonan, we selected AtGAUT1 and AtGAUT because they work together, as presented by Atmodjo et al. [48].For the estimation of gene expression, we performed molecular analyses.Firstly, we collected leaf samples (0.1 g of each sample) at 3, 7, and 21 dpi from mock and TuMVinoculated plants [120,125].Then, according to the procedure presented in [120,125], we performed RNA isolation, purification, and quality checks, as well as confirmation of lack of RNA contamination.The absence of RNA contamination was also verified again by performing reverse transcription PCR using AtEf1α and AtF-Box as reference standards [28,125], which confirmed the absence of contaminating gDNA.Then, cDNA was synthesized using the NG dART RT Kit (EURx Sp. z o.o., Gdansk, Poland), as was presented by Otulak-Kozieł et al. [28].Reverse transcription reactions were performed as described by Otulak-Kozieł et al. [125].The number of analyzed plants was selected as presented by Otulak-Kozieł et al. [28].
Real-time qPCR for analyzed genes was performed according to procedure and with the use of the equipment described by Otulak-Kozieł et al. [125] and Otulak-Kozieł et al. [28] for AtEf1α and AtF-Box as reference genes.All qPCR tests were calibrated using previously prepared 6-point calibration curves (based on cDNA and gDNA).The analyzed AtPME3, AtPME17, AtPMEI2, AtPMEI3, AtGAUT1, and AtGAUT7 were analyzed by qPCR in comparison to reference genes.The expression of these genes in A. thaliana was checked, and complete sequences were acquired from the TAIR database [126].Based on previously published papers, the primers were chosen only for AtPME17 [44]; the rest of the primers were designed using Primer3 software (version 0.4.0;Primer3Plus, Free Software Foundation, Inc., Boston, MA, USA).All the primers used during analyses are presented in Table S1.The starting cDNA solution (used for generating calibration curves) was prepared as presented by Otulak-Kozieł et al. [28].An eightfold-diluted cDNA mix was used to construct the calibration curve for gDNA, while the subsequent calibration points were measured at four-fold dilutions in a 15 µL volume.A 5 µL solution of eight-fold-diluted cDNA mix was added to the reaction mixture.The exact conditions for qPCR analyses are shown in Table S2.The expression was analyzed statistically at selected time intervals for all plants by ANOVA.

Transmission Electron Microscope (TEM) Ultrastructural Analyses of Mock and
TuMV-Inoculated Col-0, rbohD, rbohF, and rbohD/F Leaves Fragments of leaves from mock/TuMV-inoculated Col-0, rbohD, rbohF, and rbohD/F plants at 3, 7, and 21 dpi were fixed and treated according to the procedure presented in [24,120,127] for TEM.Leaf samples were collected from a total of 60 plants (30 virusinoculated and 30 mock-inoculated Col-0 and mutant plants of different types).The procedure was repeated three times, every time from a new group of plants.After TEM material preparation, the 50-70 nm sections from leaves prepared according to [24,127] were mounted on single slot copper grids from Agar Scientific (Stansted, United Kingdom: 0.75 mm Slot TEM copper Support Grids, catalog number: AGG2525C) coated with Formvar.The grids with samples were examined using transmission electron microscopy (TEM, FEI M268D "Morgagni" transmission electron microscope), as presented in [24,25], to check and characterize ultrastructural changes in the leaf cell wall.The observation in TEM was performed with a beam energy of 80 keV and snap exposure of 1.5 with the use of a SIS Morada digital camera (Olympus-SIS), and iTEM software version 5.0 (Olympus-SIS, Münster, Germany) to capture the images.

Bioinformatics Prediction of Subcellular Localization of PME3, PMEI2, PEMI3, and GAUT1 Proteins Based on Combined Predictor Database SUBA5 Search
The proteins selected for immunogold analyses were analyzed bioinformatically based on the online predictor SUBA5 [128] program (Table S3).SUBA5, as a group of online tools, enables the prediction of subcellular localization of proteins from A. thaliana based on different bioinformatic predictors.It also connects most protein predictor localization systems and new data from a mass spectrometry assay (MS/MS) gathered from the PubMed publication database [129] and integrates it into a suggested localization, the SUBA consensus (SUBAcon) [128,130].The SUBAcon parameter only suggests a major/dominant place for localization but is coanalyzed with other predictors because most proteins are mobile in plant cells and could be also localized during in-cell transportation.For this complex prediction, we selected proteins whose genes significantly changed in reaction to the TuMV infection.4.5.Quantified Immunogold Localization of PME3, PMEI2, PMEI3, GAUT1, and Two Types of HGs in Mock and TuMV-Inoculated Col-0, rbohD, rbohF, and rbohD/F Leaves Samples prepared according to the TEM procedure as presented in Section 4.3 were also used for the preparation of 50-70 nm sections from leaves of all analyzed plants for immunogold localization.This section was next mounted to single-slot nickel grids from Agar Scientific (Stansted, United Kingdom: 0.75mm Slot TEM nickel Support Grids, catalog number: AGG2525N) coated with Formvar and treated according to the immunogold labeling procedure presented previously [24,125].The immunogold labeling was performed to separately detect proteins PME3, PMEI2, PMEI3, and GAUT1, and two types of low/unesterificated HG (homogalacturonan) and highly methylesterificated HG.The selection of localized elements was made based on the results of gene expression and the bioinformatic analyses of the prediction of subcellular localization in SUBA5.For the detection of PME3, PMEI2, PMEI3, GAUT1, two types of low/unesterificated HG (homogalacturonan), and highly methylesterificated HG, we used different types of primary polyclonal/monoclonal antibodies targeting the selected elements.For the detection of PME3 (NCBI and TAIR protein numbers: NP_188048.1,AT3G14310.1),PMEI2 (NCBI and TAIR protein numbers: NP_188348.1,AT3G17220.1),and PMEI3 (TAIR protein numbers: AT5G20740.2),we used three custom-designed GeneCust (Boynes, France) polyclonal rabbit antibodies.Antibodies were designed separately for highly immunogenic C-terminal parts of PME3 (QGSGVKADATVAADGSGTFK), PMEI2 (GPSTCEQDMADFKVDPSA), and PMEI3 (NQLDETRGKPHDVHL).Sequences for designing were downloaded from NCBI and TAIR databases [126,131].For the detection of GAUT1, we used rabbit polyclonal antibodies manufactured by PhytoAB Inc. (San Jose, CA, USA, catalog number: PHY1047S).In the case of low/unesterificated HG and highly methylesterificated HG, we used monoclonal rat antibodies JIM5 and JIM7, respectively, from the laboratory of Paul Knox, Ph.D., University of Leeds, United Kingdom, currently distributed by Agrisera (Vänäs, Sweden, catalog numbers: AS18 4194-1ml AS18 4195-1ml).The primary antibodies for immunogold localization were used in a 1:50 dilution in the case of the polyclonal rabbit antibodies (PME3, PMEI2, and PEMI3) and a 1:10 dilution in the case of the monoclonal rat antibodies (JIM5 and JIM7).Visualization was performed using secondary antirabbit or antirat antibodies conjugated with 18 nm nanogold particles (Jackson ImmunoResearch Europe Ltd., Cambridgeshire, UK, catalog numbers: 711-215-152, 112-215-143).The immunogold-labeled sections on the nickel grids were examined using a transmission electron microscope [24,26] with the parameters described in Section 4.3.Then, the localization of selected proteins and different types of HG was quantified following the method of Luschin-Ebengreuth and Zechmann [132] globally in the case of mock-inoculated and TuMV-inoculated plants.Statistical analyses were performed as described by Otulak-Kozieł et al. [24].The concentrations of gold particles globally were validated using ANOVA and post hoc Tukey's HSD (honest significant difference) test using Statistica software (version 13.0; StatSoft and TIBCO Software Inc., Palo Alto, CA, USA).The statistical estimation (with use of ANOVA) of immunogold labeling was performed for virus-inoculated and mockinoculated samples and compared at 7 and 21 dpi (PME3, PMEI2, PMEI3, and GAUT1) and 21 dpi (low/unesterificated HG and highly methylesterificated HG).The number of gold particles globally was counted in 35 fields (10 µm 2 ) per image.For each combination (mock-inoculated plants and TuMV-inoculated Col-0, rbohD, rbohF, and rbohD/F plants), gold particles from 200 photographs were counted to determine the presence of different proteins or HG.To validate the activity of PME, soluble protein extracts were generated from mockand virus-inoculated leaves from Col-0, rbohD, rbohF, and rbohD/F plants from 7 and 21 dpi.For each combination, three replicates of extracts were generated.The leaf tissue from mock-inoculated and virus-inoculated plants was ground in liquid nitrogen and added to twice the fresh weight (w/v) of extraction buffer (100 mM Tris-HCl, pH 7.5, 500 mM NaCl) containing a protease inhibitor cocktail (ABMGood, Richmond, BC, Canada, catalog number: G135), as was described by Müller et al. [133] and Grsic-Rausch and Rausch [134].Extracts from leaves were then rotated at 4 • C for 30 min and centrifuged at 11,500× g at 4 • C for 20 min.Fresh supernatants were used immediately for all enzyme assays, as presented by Müller et al. [133].A coupled enzymatic assay was performed as described by Grsic-Rausch and Rausch (2004) using a spectrophotometric plate reader.For this purpose, we used Agilent BioTech Epoch 121221F, (Agilent, Santa Clara, CA, USA).The plate reader records the changes in absorption at 340 nm over 15 min at room temperature.The change in absorption per unit time over the linear part of the reaction was calculated for each well and used to calculate the increase in concentration of NADH.The NADH concentration was calculated using the extinction coefficient ε 340 for NADH (6220 M −1 cm −1 ).PME activity was defined as U, where 1 U is 1 nmol of converted NADPH/s/mg total protein.The results were analyzed statistically using ANOVA and post hoc Tukey's HSD (honest significant difference) test using Statistica software (version 13.0; StatSoft and TIBCO Software Inc., Palo Alto, CA, USA).To validate methylesters in the cell wall from mock-and virus-inoculated leaves of Col-0, rbohD, rbohF, and rbohD/F were then ground in liquid nitrogen, and 200 mL of methanol was added, as was previously described by Müller et al. [135].The ground leaf tissue was extracted four times with a 1:1 (v/v) methanol:chloroform mixture, washed once with acetone, and dried overnight at room temperature.The weight of the dried cell wall materials was determined, and 0.5-1.0mg was washed with 2 mL of water.To release the methylesters from the cell wall, the material was incubated for 1 h at room temperature with 100 mL of 0.5 M NaOH.After neutralization with 50 mL of 1 M HCl, the samples were centrifuged at 2000× g for 10 min.A generated supernatant was used to quantify the methanol, which was released during saponification according to the procedure presented by Klavons and Bennett [136] and Müller et al. [135] to generate extract from mock-and virus-inoculated leaves of Col-0, rbohD, rbohF, and rbohD/F.In the final step, the absorbance was measured at 412 nm using an Agilent BioTech Epoch 121221F (Agilent, USA) and compared with a standard curve generated with a methanol dilution series, as was presented by Müller et al. [135].The results were analyzed statistically with the use of ANOVA and post hoc Tukey's HSD (honest significant difference) test using Statistica software (version 13.0; StatSoft and TIBCO Software Inc., Palo Alto, CA, USA).Estimated methylester content was based on the methanol associated with saponified cell wall materials in leaves.Therefore, the estimated methylester content was presented as nmol methanol/mg cell wall material, as was suggested by Müller et al. [135].

Conclusions
These studies highlighted complex and dynamic cell wall modifications concentrated on selected elements/molecules associated with pectin metabolism during NADPH oxidase homologs D and F deficient A. thaliana mutants' reaction to TuMV.Our findings indicated the importance of selected PME and PMEIs in regulating cell wall changes in Arabidopsis rboh-TuMV infections.The rbohF-TuMV and especially rbohD/F-TuMV mutant interactions displayed virus content limitation and dynamic cell wall rebuilding in all A. thaliana leaf tissues along with induced PMB formation or phenolic-like compound deposition, especially in vascular bundles.In contrast, Col-0 and rbohD mutants promoted TuMV infection and characterized cell wall rearrangement with induction of multivesicular structures and thickening in the plasmodesmata area in mesophyll and vascular tissue.As we confirmed previously [27], RbohF can promote increased susceptibility against TuMV.These susceptible reactions were displayed with upregulation of AtPME3, in contrast to AtPME17, which was also confirmed by induction of PME3 deposition.Our results revealed the highest PME activity in rbohD-TuMV, as well as a decrease in cell wall methylesters compared to mock-inoculated plants in both resistance interactions.Consequently, ultrastructural validation with quantification indicated that the susceptible reaction of rbohD and Col-0 to TuMV was characterized by significant domination of low/non-methylesterificated HGs.Conversely, cell wall rebuilding in the resistance response of rbohF and rbohD/F to TuMV was associated with dynamic induction of AtPMEI2 and AtPMEI3, and also AtGAUT1 with AtGAUT7, which was additionally confirmed by significant induction of the deposition of PMEI2, PMEI3, GAUT1, and GAUT7 proteins.Therefore, PMEI2 and PMEI3 can be important signaling resistance factors in the rboh-TuMV pathosystem.Cell wall changes in both resistance reactions were an effect of an intense decrease of PME activity, which was the most intense in rbohD/F-TuMV.It was accompanied by the induction of cell wall methylester content.Consequently, in contrast to susceptible reaction, dominated highly methylesterificated HGs were actively distributed while participating in rbohF and rbohD/F defense response and cell wall rebuilding.These results may help to provide new information on better understanding the mechanisms of defense response to TuMV.Further studies are needed to elucidate the confirmation of other components of cell wall structures participating in apoplast rearrangements in NADPH oxidase homologs D-and F-deficient mutants-the TuMV pathosystem.

Figure 2 .
Figure 2. Normalized relative expression levels of AtPME3 (A), AtPME17 (B), AtPMEI2 (C), AtPEMI3 (D), AtGAUT1 (E), and AtGAUT7 (F), calculated based on the mean expression of AtEf1α and AtF-Box reference genes in mock-inoculated and virus-inoculated Col-0, rbohD, rbohF, and rbohD/F plants at 3, 7, and 21 dpi.The mean values of the normalized expression levels were calculated and analyzed using ANOVA and Tukey's HSD test at p < 0.05.Statistically significant values are indicated by different letters above the bars.

Figure 7 .
Figure 7. Estimation of PME activity in mock-inoculated and virus-inoculated Col-0, rbohD, r and rbohD/F leaves at 7 and 21 dpi.The mean activities (in U/mg total protein) were calcul Significant differences between classes at the p < 0.05 level were assessed by ANOVA with pos Tukey's HSD.Statistically significant values are indicated by letters above the chart bars.

Figure 7 .
Figure 7. Estimation of PME activity in mock-inoculated and virus-inoculated Col-0, rbohD, rbohF, and rbohD/F leaves at 7 and 21 dpi.The mean activities (in U/mg total protein) were calculated.Significant differences between classes at the p < 0.05 level were assessed by ANOVA with post hoc Tukey's HSD.Statistically significant values are indicated by letters above the chart bars.

Figure 7 .
Figure 7. Estimation of PME activity in mock-inoculated and virus-inoculated Col-0, rbohD, rbohF, and rbohD/F leaves at 7 and 21 dpi.The mean activities (in U/mg total protein) were calculated.Significant differences between classes at the p < 0.05 level were assessed by ANOVA with post hoc Tukey's HSD.Statistically significant values are indicated by letters above the chart bars.

Figure 8 .
Figure 8.Quantification of methylesters in mock-inoculated and virus-inoculated Col-0, rbohD, rbohF, and rbohD/F leaves at 7 and 21 dpi.Estimated methylester content as based on methanol associated with saponified cell wall materials in leaves.Significant differences between classes at the p < 0.05 level were assessed by ANOVA with post hoc Tukey's HSD.Statistically significant values are indicated by letters above the chart bars.