Rory N. Pruitt, Benjamin Schwessinger, Anna Joe, Nicholas Thomas, Furong Liu, Markus Albert, Michelle R. Robinson, Leanne Jade G. Chan, Dee Dee Luu, Huamin Chen, Ofir Bahar, Arsalan Daudi, David De Vleesschauwer, Daniel Caddell, Weiguo Zhang, Xiuxiang Zhao, Xiang Li, Joshua L. Heazlewood, Deling Ruan, Dipali Majumder, Mawsheng Chern, Hubert Kalbacher, Samriti Midha, Prabhu B. Patil, Ramesh V. Sonti, Christopher J. Petzold, Chang C. Liu,

Jennifer S. Brodbelt, Georg Felix, Pamela C. Ronald

CROP SCIENCE, Published 24 July 2015, Sci. Adv. 1, e1500245 (2015)

DOI: 10.1126/sciadv.1500245 (www.advances.sciencemag.org/cgi/content/full/1/6/e1500245/DC1 )

ABSTRACT

Surveillance of the extracellular environment by immune receptors is of central importance to eukaryotic survival. ThericereceptorkinaseXA21,whichconfers robust resistance to most strains of the Gram-negative bacterium Xanthomonas oryzae pv. Oryzae (Xoo), is representative of a large class of cell surface immune receptors in plants and animals. We report the identification of a previously undescribed Xoo protein, called RaxX, which is required for activation of XA21-mediated immunity.Xoo strains that lack RaxX, or carry mutations in the single RaxX tyrosine residue (Y41), are able to evade XA21-mediated immunity. Y41 of RaxX is sulfated by the prokaryotic tyrosine sulfotransferase RaxST. Sulfated, but not nonsulfated, RaxX triggers hallmarks of the plant immune response in an XA21-dependent manner. A sulfated, 21–amino acid synthetic RaxX peptide (RaxX21-sY) is sufficient for this activity. Xoofield isolates that overcome XA21-mediated immunity encode an alternate raxXallele, suggesting that co-evolutionary interactions between host and pathogen contribute to RaxX diversification. RaxX is highly conserved in many plant pathogenicXanthomonasspecies. The new insights gained from the discovery and characterization of the sulfated protein, RaxX, can be applied to the development of resistant crop varieties and therapeutic reagents that have the potential to block microbial infection of both plants and animals.

Fig. 1. raxX, a small ORF located upstream of theraxSTABoperon, is required for activation of XA21-mediated immunity.(A) raxST, raxA,and raxB are encoded in a single operon. A 1.0-kb region upstream ofraxST and a 1.7-kb region downstream ofraxB were deleted in PXO99D1.0

Sp and PXO99D1.7 Sp, respectively. The raxXORF is located ~0.4 kb upstream ofraxSTand in the opposite orientation. (B) TP309 (open bars) or XA21-TP309 (black bars) were inoculated by clipping with scissors dipped inXoosuspensions. Bars indicate the mean lesion length ± SE measured 14 days after inoculation (n≥14). The“*”indicates statistically significant difference from PXO99 using Dunnett’stest(a= 0.01). No statistical differences in lesion length were observed on TP309 inoculated with the different strains. The experiment was repeated at least three times with similar results. (C) XA21-TP309 rice leaves display water-soaked lesions 2 weeks after inoculation with the indicated strains. (D) GrowthofPXO99 (□), PXO99DraxX(△), and PXO99DraxX (praxX)(○) in rice leaves inoculated as in (B). In planta bacterial growth analysis was carried out as described (22). Bacterial quantification was determined as the number of colony-forming units (CFU) extracted per inoculated leaf. For the final data point,“*” indicates statistically significant difference from PXO99 using

Dunnett’stest(a= 0.01,n= 4). The experiment was repeated twice with similar results.