D ISCUSSION

In spite of recent advances on chitin perception in non-legumes, such as A. thaliana and rice, little is known about chitin-dependent defense activation in legumes, which are hosts of LCO-deploying endosymbioses. In this work, we developed an experimental system to evaluate the capacity of chitin to induce defense reactions in roots of M. truncatula by, first, preparing from crab shell chitin a purified chitooligosaccharide fraction containing high- DP COs, and second, establishing a standardized elicitation assay for medium to high- throughput applications.

The first steps of our procedure to prepare COs were derived from established protocols for purification and acid hydrolysis of CSC (Shahidi and Abuzaytoun 2005), followed by low performance gel filtration chromatography. The obtained fractions were characterized by solid, as well as liquid state NMR analysis. Compared to other techniques, solid state NMR analysis is truly quantitative and not sensitive to either response factors (as in mass spectrometry), or calibration (as in gel filtration chromatography), and does not need to dissolve the sample. Following our procedure, we were able to generate from 1 g CSC more than 10 mg of CO fractions of average DP 13.7 (CO I) and 6.8 (CO II), as evaluated by solid state RMN, which were 97 and 100% acetylated, respectively. Both fractions were thus promising as potential abundant sources of bioactive high-DP COs. However, we observed a surprising discrepancy between the DP values estimated from solid state NMR (13.7) and liquid state NMR (2.7) for the CO I fraction. This result is likely related to the ability of -1,4-linked oligosaccharides to build intermolecular associations, resulting in irreversible aggregation and insolubility. After gel filtration and drying, the high-DP COs in CO I were no longer soluble, and were thus not accessible to liquid state NMR analysis anymore, yet still accessible to solid state NMR analysis, which resulted in diverging average DP estimations between the two methods. This observation illustrates the general difficulty of characterizing COs of relatively high DP and the impact of the drying protocol on the dissolution properties. To our knowledge, these aggregation issues have never been demonstrated so far for chitin oligosaccharides, and our data suggest that the concentrations of CO mixtures that have been used in many studies on chitin elicitation, ranging from 100 µg.ml^-1 to 1 mg.ml^-1, might have been be overestimated with regards to the actual concentration and DP of COs in solution. We thus recommend selecting the

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fraction of apparent molecular weight between 2 and 3 kDa (CO II) to prepare the required CO elicitor.

Not only the actual concentration of elicitors, but also their purity, are essential parameters when studying their perception by plants. It was recently shown that COs were contaminating a laminarioligosaccharide preparation, causing confused interpretation of its elicitor activity (Desaki, et al. 2011). We selected for further characterization the fully acetylated CO II preparation with good dissolution properties and a mean DP of 6.8 (COs DP7), very likely containing a high proportion of COs of DP7 and 8 that were shown to be most active in the literature (Day, et al. 2001; Shibuya and Minami 2001; Zhang, et al. 2002).

In order to prove the absence of any compound able to elicit chitin-independent defense reactions, this preparation was tested on the cerk1 mutant of A. thaliana at 100 µg.ml^-1, a concentration usually employed for elicitation of A. thaliana by CO mixtures (Ramonell, et al.

2005; Zhang, et al. 2002). In contrast to WT seedlings, mutant seedlings were unable to produce ROS in response to COs DP7, and all the genes that responded to COs DP7 in WT seedlings lost their responsiveness in cerk1 seedlings (Fig. 2).The COs DP7 preparation was therefore validated as being essentially free of contaminating non-CO elicitors. It was then retained to address its activity in M. truncatula roots, using a standardized bioassay that we developed in the frame of this study.

Unlike A. thaliana, M. truncatula seedlings cannot be cultivated submerged.

However, the use of moderate amounts of liquid medium in small petri dishes gave rise after one week to the development of seedlings with upright hypocotyls and immersed roots, which were responsive to the treatment with a well-defined elicitor of defense responses in legumes, the -glucan from P. sojae (Ayers, et al. 1976). All but one of a set of defense- associated genes, encoding pathogenesis-related (PR) proteins, enzymes involved in the redox homeostasis, and in the biosynthesis of isoflavonoid phytoalexins, were validated as elicitation marker genes under our conditions, being upregulated in response to the control elicitor, in a pattern similar to the one reported for M. truncatula cell cultures treated with the pure hepta--glucoside or a proteinaceous elicitor of yeast (Leclercq, et al. 2008). In addition, our experimental set up allowed measuring non-destructively the early production of extracellular ROS by analyzing small aliquots of the extracellular medium, until the harvest of the root system for destructive measurement of gene expression.

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Using our bioassay, we showed that COs DP7 induced an oxidative burst (Fig. 4) and defense markers (Fig. 5) in M. truncatula. The perception system for COs of M. truncatula was found to be very sensitive, since concentrations as low as 0.02 µg.ml^-1 COs DP7, which corresponds to approximately 50 nM chitoheptaose equivalent concentration, induced the production of H2O2, while 2 µg.ml^-1 COs DP7 resulted in approximately half-maximal induction of the oxidative burst (Fig. 4b). Considering the yield of the COs DP7 preparation and the sensitivity of our assay, it can be calculated that 1 g of crab shell chitin allows the preparation of COs in amounts sufficient to elicit 2160 seedlings with 20 µg.ml^-1 of elicitor.

As observed for A. thaliana (Ramonell, et al. 2002), the up-regulation of chitin-induced genes was rapid but transient in M. truncatula roots, declining after 1 h treatment for most of the genes of our selection, which were even slightly down-regulated at 12 h (Fig. 5). This data shows on the one hand the importance of selecting early time points to assess CO elicitor activity in M. truncatula roots. On the other hand, the GLP gene marker from our selection might be of special interest because of its sustained induction upon CO treatment.

Our biological assay will prove useful each time it is required that roots are readily accessible for treatment or for measurement of physiological responses, for example for the deciphering of the perception mechanisms and signaling pathways involved in responses of M. truncatula roots to signals from soil-borne microorganisms. As a proof of concept, we applied culture filtrates of A. euteiches, an important soil-borne pathogen of pea, to M.

truncatula roots grown in the hydroponic test system. The induction of the production of H2O2 strongly suggested the presence of elicitor-active compounds in the culture filtrate, which were released into the medium by the growing oomycete (Fig. 6). The application of the M. truncatula root assay will enable the bioactivity-based purification of these elicitor- active compounds, which might include chitosaccharidic fragments released from the cell wall surface (Badreddine, et al. 2008).

In conclusion, we provide a rigorous procedure to generate and to purify ample amounts of a highly active chitooligosaccharide elicitor that combines the presence of fully acetylated COs of DP higher than 6 with good solubility properties. Our procedure is accessible to most laboratories and represents a valuable alternative to commercial CO products, which are either poorly characterized, or are devoid of COs of DP higher than 6, or are expensive when they grant the presence of a pure high-DP CO. On this occasion, we illustrate how the dissolution properties of COs can be studied by a combination of liquid-

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state and solid-state NMR and we show that high-DP range CO fragments aggregate after lyophilization, a property that might cause inconsistent results when exploring their biological activity. In addition, we have established a fast and versatile standardized elicitation assay for M. truncatula, and show that its root system responds with high sensitivity, but transiently, to the CO elicitor. This assay will be fundamental to the identification of potential chitosaccharide elicitors from A. euteiches. It may also be of value for studies of other physiological responses of M. truncatula roots, such as responses to salt stress or drought.

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