Abstract
SUGARWINs are PR-4 proteins associated with sugarcane defense against phytopathogens. Their expression is induced in response to damage by Diatraea saccharalis larvae. These proteins play an important role in plant defense, in particular against fungal pathogens, such as Colletothricum falcatum (Went) and Fusarium verticillioides. The pathogenesis-related protein-4 (PR-4) family is a group of proteins equipped with a BARWIN domain, which may be associated with a chitin-binding domain also known as the hevein-like domain. Several PR-4 proteins exhibit both chitinase and RNase activity, with the latter being associated with the presence of two histidine residues H11 and H113 (BARWIN) [H44 and H146, SUGARWINs] in the BARWIN-like domain. In sugarcane, similar to other PR-4 proteins, SUGARWIN1 exhibits ribonuclease, chitosanase and chitinase activities, whereas SUGARWIN2 only exhibits chitosanase activity. In order to decipher the structural determinants involved in this diverse range of enzyme specificities, we determined the 3-D structure of SUGARWIN2, at 1.55Å by X-ray diffraction. This is the first structure of a PR-4 protein where the first histidine has been replaced by asparagine and was subsequently used to build a homology model for SUGARWIN1. Molecular dynamics simulations of both proteins revealed the presence of a flexible loop only in SUGARWIN1 and we postulate that this, together with the presence of the catalytic histidine at position 42, renders it competent as a ribonuclease. The more electropositive surface potential of SUGARWIN1 would also be expected to favor complex formation with RNA. A phylogenetic analysis of PR-4 proteins obtained from 106 Embryophyta genomes showed that both catalytic histidines are widespread among them with few replacements in these amino acid positions during the gene family evolutionary history. We observe that the H11 replacement by N11 is also present in two other sugarcane PR-4 proteins: SUGARWIN3 and SUGARWIN4. We propose that RNase activity was present in the first Embryophyta PR-4 proteins but was recently lost in members of this family during the course of evolution.
Highlights
Plant responses to insects and pathogens are complex and modulate the expression of a large number of genes, many of which are believed to have a direct role in plant defense (Xu et al, 1994; Agrawal et al, 2003; Banno et al, 2005; Franco et al, 2017)
Among the 436 PR-4 protein sequences found in our searches, 312 and 101 of them had histidine or asparagine at position 11 [H42—SUGARWIN1; N37— SUGARWIN2; N43—SUGARWIN3; N38—SUGARWIN4], respectively, whereas 362 and 18 had histidine or asparagine at position 113 [H142—SUGARWIN1; H135—SUGARWIN2; N142—SUGARWIN3; H137—SUGARWIN4] (Figure 1)
We suggest that SUGARWIN1 and other ribonuclease PR-4 proteins must provide a significant flexibility at the active site to assure the catalytic histidine properties for catalysis
Summary
Plant responses to insects and pathogens are complex and modulate the expression of a large number of genes, many of which are believed to have a direct role in plant defense (Xu et al, 1994; Agrawal et al, 2003; Banno et al, 2005; Franco et al, 2017). Pathogen recognition by plants activates the host defense response resulting in the accumulation of pathogenesis-related proteins (PR proteins) (Pieterse and van Loon, 1999). PR-4 proteins composed only by BARWIN domains were identified in several plants, such as: tobacco (Friedrich et al, 1991), tomato (Linthorst et al, 1991), Arabidopsis (Potter et al, 1993), wheat (Caruso et al, 1999), W. japonica (Kiba et al, 2003), maize (Bravo et al, 2003), rice (Agrawal et al, 2003; Zhu et al, 2006) and L. radiata (Li et al, 2010). The PR-4 proteins are grouped into class I and class II based on the presence or absence of this chitin-binding domain (Neuhaus et al, 1996)
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