Xanthomonas oryzae pv. oryzae XopQ protein suppresses rice immune responses through interaction with two 14‐3‐3 proteins but its phospho‐null mutant induces rice immune responses and interacts with another 14‐3‐3 protein

Abstract

SummaryMany bacterial phytopathogens employ effectors secreted through the type‐III secretion system to suppress plant innate immune responses. The Xanthomonas type‐III secreted non‐TAL effector protein Xanthomonas outer protein Q (XopQ) exhibits homology to nucleoside hydrolases. Previous work indicated that mutations which affect the biochemical activity of XopQ fail to affect its ability to suppress rice innate immune responses, suggesting that the effector might be acting through some other pathway or mechanism. In this study, we show that XopQ interacts in yeast and in planta with two rice 14‐3‐3 proteins, Gf14f and Gf14g. A serine to alanine mutation (S65A) of a 14‐3‐3 interaction motif in XopQ abolishes the ability of XopQ to interact with the two 14‐3‐3 proteins and to suppress innate immunity. Surprisingly, the S65A mutant gains the ability to interact with a third 14‐3‐3 protein that is a negative regulator of innate immunity. The XopQS65A mutant is an inducer of rice immune responses and this property is dominant over the wild‐type function of XopQ. Taken together, these results suggest that XopQ targets the rice 14‐3‐3 mediated immune response pathway and that its differential phosphorylation might enable interaction with alternative 14‐3‐3 proteins.

Interaction of theXanthomonaseffectors XopQ and XopX results in induction of rice immune responses

“…In order to check if XopX would interact with any of the rice 14-3-3 proteins, we cloned the wild-type xopX gene in the pDEST32 vector yielding the BD∷xopX (DNA-Binding Domain) clone (as listed in Supplementary table S2). We screened xopX against the eight rice 14-3-3 proteins cloned in the pDEST22 vector yielding the AD∷gf14a-h (Activation Domain) clones used in an earlier study (Deb et al, 2019) (listed in Supplementary table S2), using the yeast two-hybrid system and the yeast strain pJ694a (James et al, 1996). The one-to-one yeast two-hybrid screen indicated that the XopX protein showed physical interaction with two of the eight rice 14-3-3 proteins, Gf14d and Gf14e (Fig 1A).…”

“…The wild-type copy of the xopX gene and its 14-3-3 protein binding motif mutants were cloned in the yeast two-hybrid vector pDEST32 (Invitrogen) using the Gateway cloning system (Invitrogen, California). The eight rice 14-3-3 genes cloned in the yeast two-hybrid vector pDEST22 (Invitrogen) were used from a previous study (Deb et al, 2019). For analysis of interaction of xopX with the other effector proteins, the pDEST22 clones containing xopN , xopQ and xopZ were used, whereas xopX was cloned in pDEST32.…”

“…For example, the Xanthomonas oryzae pv. oryzae effector XopQ interacts with the rice 14-3-3 proteins to suppress immune responses (Deb et al, 2019). The AvrPto effector has also been shown to interact with the plant receptor FLS2, which recognizes the bacterial flagellin, further suppressing host PTI (Xiang et al, 2008).…”

“…The XopQ protein is highly conserved in Xanthomonads (Hajri et al, 2009, Moreira et al, 2010, Jalan et al, 2013, Potnis et al, 2011), and has been shown to require its biochemical activity for complete virulence in rice (Gupta et al, 2015). XopQ also has motifs for binding to 14-3-3 proteins (Dubrow et al, 2018, Deb et al, 2019). The 14-3-3 proteins are eukaryotic adapter proteins which play key roles in multiple cellular events in plants (Cotelle and Leonhardt, 2015, Cotelle et al, 2000).…”

“…The ability of X. oryzae pv. oryzae XopQ protein to interact with 14-3-3 proteins has been shown to be important for its ability to suppress rice immune responses (Deb et al, 2019). The X. campestris pv.…”