Role of salicylic acid in the response of B. napus to V. longisporum

Plant derived phenylpropanoids are the most common secondary metabolic compounds in plants, bacteria and fungi, many of which are used in the protection against biotic or

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abiotic stresses or play essential roles in plant physiology (Seigler 1999; Korkina et al.

2011). Phenols bear a 3-carbon chain attached to a 6-carbon aromatic ring and commonly are formed from t-cinnamic or p-coumaric acids. Among these components, simple phenolic acids and esters, flavonoids and lignin have been reported to be functional in the resistance of various host plants against Verticillium pathogens or have direct effects on fungal growth (Howell et al. 1976; Picman et al. 1995; Debode et al. 2005; Eynck et al.

2009a; Xu et al. 2011; König et al. 2014). In cotton, enzyme activities of PAL and POX were increased after inoculation with V. dahliae (Xu et al. 2011). Furthermore, lignin is believed to contribute to the resistance of cotton to V. dahliae according to the observation of more intensive lignification in the resistant cultivar. Besides, salicylic acid (SA) has been reported to be involved in disease defense as well.

Similar to tomato and tobacco, a remarkable increase of susceptibility to V. longisporum was observed in the present study when using SA-deficient transgenic NahG oilseed rape, indicating an essential role of SA in basal resistance of oilseed rape against V. longisporum (Achuo et al. 2004). As described in previous studies, SA can be transported through phloem to all plant parts as a mobile signal triggering local and systemic acquired resistance against viruses or biotrophic pathogens. In addition, it may be accumulated in xylem vessels by root colonized soil-borne or seed-borne pathogens and may be long-distance transported toward distal parts of the plants for response to disease (Ben-Tal and Cleland 1982; Rocher et al. 2006; Ratzinger et al. 2009; Rivas-San Vicente and Plasencia 2011). The level of conjugated SA found in wild type oilseed rape is at least 4-fold higher than free SA, which is believed to be actively transported from the cytosol into the vacuole for storage and released as free SA when necessary to trigger systemic acquired resistance during infection (Rivas-San Vicente and Plasencia 2011).

Infection of V. longisporum caused induction of both conjugated and free SA in oilseed rape, but only conjugated SA and no free SA was accumulated after exogenous application of SA. This result proved that oilseed rape prefers to convert free SA into the conjugated form preventing phytotoxic effects similar to A. thaliana (Wildermuth 2006;

Nobuta et al. 2007), since an exogenously applied higher concentration of SA was confirmed in the present study to be phytotoxic to host plants as well. In contrast to enhancing the resistance of plants against other pathogens (Chen et al. 2016), an exogenous application of 0.5 mM SA is not efficient to reduce infection of V. longisporum on oilseed rape. In the present study, no effect was observed on the growth of V. longisporum up to a concentration of 1.5 mM exogenously applied SA, which is substantially higher than the endogenous SA levels in diseased plants which have been reported to be in a range from 4 to 80 µg/g fresh weight in previous studies (Ratzinger et

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al. 2009; Kamble et al. 2013). Since the reported concentration of SA in V. longisporum infected plant tissues did not inhibit fungal growth in vitro but showed a weak to moderate promoting effect, SA is unlikely to suppress the growth of pathogens in plants as a antifungal compound but plays a role in signaling as trigger or modulator (Rüffer et al.

1995).

The first penetration of roots by V. longisporum has been reported to appear after 60 hpi in plants root-dip inoculated with a conidia suspension (Eynck et al. 2007). In the early response of B. napus to V. longisporum, induction of SA was faster in the resistant cultivar than in the susceptible cultivar. However, in contrast to A. thaliana, the quantitative resistance of oilseed rape to V. longisporum seems to be dependent on neither SA- nor JA- associated plant resistant proteins PR1, PR2 and PDF1.2 (Johansson et al. 2006b;

Kamble et al. 2013). By infection with V. longisporum, PR1 and PR2 were up-regulated.

However, higher expression of PR1 and PR2 were unexpectedly found in susceptible cultivar, which may indicate that the induction of these proteins did not enhance cultivar-related resistance of oilseed rape against V. longisporum. Similar results have been obtained by Coquoz et al. (1995) on potato: the exogenous application of SA on potato led to an increase in PR gene expression but not the resistance to Phytophthora infestans and Alternaria solani. However, the gene expression to unit of pathogen amount showed another image, that stronger upregulation towards fungus was found in resistant cultivar.

The responses relative to fungal biomass can more relevant to accurately describe plant responses tp a certain unit of pathogen biomas. In contrast to A. thaliana, in which the accumulation of SA induced by pathogen or elicitor is synthesized through the isochorismate pathway, a similar tendency of enhanced enzyme activity of benzoic acid 2-hydroxylase (BA2H) and content of SA was observed in the present study indicating that V. longisporum-induced accumulation of SA possibly involves the synthesis via phenylalanine. Interestingly, an identical induction of SA has not only been observed in the resistant but also in the susceptible cultivar. However, the induction in the susceptible cultivar was delayed and first observed only at 14 dpi, when the pathogen assumingly had already colonized the cortex of roots and spread into the vascular vessels (Eynck et al. 2007). A higher biomass of V. longisporum was detected as well in the hypocotyl of the susceptible cultivar at this time point. As well known, the biosynthesis of SA and lignin share the same precursors, such as t-cinnamic acid and phenylalanine. Therefore, these two biosynthetic pathways may have a competitive relationship. Phenolic acids and lignin have been shown to be important for resistance of B. napus to V. longisporum by producing occlusions in the xylem vessels for inhibiting the spread of the fungus (Eynck et al. 2009a; Kamble et al. 2013). Both resistant and susceptible cultivars tested in the

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present study showed increases of phenolic acids after infection with V. longisporum. The accumulation of phenolic acids happened at 14 dpi, while increase of SA was induced by