The barley stripe mosaic virus (BSMV) triple gene block 1 (TGB1) protein is required for virus cell-to-cell movement. However, little information is available about how these activities are regulated by post-translational modifications. In this study, we showed that the BSMV Xinjiang strain TGB1 (XJTGB1) is phosphorylated in vivo and in vitro by protein kinase CK2 from barley and Nicotiana benthamiana. Liquid chromatography tandem mass spectrometry analysis and in vitro phosphorylation assays demonstrated that Thr-401 is the major phosphorylation site of the XJTGB1 protein, and suggested that a Thr-395 kinase docking site supports Thr-401 phosphorylation. Substitution of Thr-395 with alanine (T395A) only moderately impaired virus cell-to-cell movement and systemic infection. In contrast, the Thr-401 alanine (T401A) virus mutant was unable to systemically infect N. benthamiana but had only minor effects in monocot hosts. Substitution of Thr-395 or Thr-401 with aspartic acid interfered with monocot and dicot cell-to-cell movement and the plants failed to develop systemic infections. However, virus derivatives with single glutamic acid substitutions at Thr-395 and Thr-401 developed nearly normal systemic infections in the monocot hosts but were unable to infect N. benthamiana systemically, and none of the double mutants was able to infect dicot and monocot hosts. The mutant XJTGB1T395A/T401A weakened in vitro interactions between XJTGB1 and XJTGB3 proteins but had little effect on XJTGB1 RNA-binding ability. Taken together, our results support a critical role of CK2 phosphorylation in the movement of BSMV in monocots and dicots, and provide new insights into the roles of phosphorylation in TGB protein functions.

Nucleotide-binding leucine-rich repeat (NLR) receptor-mediated immunity includes rapid production of reactive oxygen species (ROS) and transcriptional reprogramming, which is controlled by transcription factors (TFs). Although some TFs have been reported to participate in NLR-mediated immune response, most TFs are transcriptional activators, and whether and how transcriptional repressors regulate NLR-mediated plant defenses remains largely unknown. Here, we show that the Alfin-like 7 (AL7) interacts with N NLR and functions as a transcriptional repressor. Knockdown and knockout of AL7 compromise N NLR-mediated resistance against tobacco mosaic virus, whereas AL7 overexpression enhances defense, indicating a positive regulatory role for AL7 in immunity. AL7 binds to the promoters of ROS scavenging genes to inhibit their transcription during immune responses. Mitogen-activated protein kinases (MAPKs), salicylic acid-induced protein kinase (SIPK), and wound-induced protein kinase (WIPK) directly interact with and phosphorylate AL7, which impairs the AL7-N interaction and enhances its DNA binding activity, which promotes ROS accumulation and enables immune activation. In addition to N, AL7 is also required for the function of other Toll interleukin 1 receptor/nucleotide-binding/leucine-rich repeats (TNLs) including Roq1 and RRS1-R/RPS4. Our findings reveal a hitherto unknown MAPK-AL7 module that negatively regulates ROS scavenging genes to promote NLR-mediated immunity.