Figure 2.

CRISPR/Cas9-induced targeted knockout of SOBIR1 in transgenic N. benthamiana:Cf-4 abolishes the functionality of the Cf-4 transgene. A, Nucleotide sequence alignment of the regions in SOBIR1 (upper panel) and SOBIR1-like (lower panel) targeted by sgRNAs in the two N. benthamiana:Cf-4 double sobir1/sobir1-like knock-out lines, with wild-type SOBIR1 and SOBIR1-like sequences, respectively. The sgRNA sequences are shown in orange, and the PAM sites are indicated in blue. The deleted nucleotides in the generated transformant are indicated with carmine dashes, and the inserted nucleotides are denoted with carmine letters. The type of mutations and the numbers of deleted/inserted nucleotides are shown on the right. B, Nucleotide sequence alignment of the regions in SOBIR1 (upper panel) and SOBIR1-like (lower panel) targeted by sgRNAs in the N. benthamiana:Cf-4 single sobir1 knock-out line, with wild-type SOBIR1 and SOBIR1-like sequences, respectively. The sgRNA sequences are shown in orange, and the PAM sites are indicated in blue. The deleted nucleotides in the generated transformant are indicated with carmine dashes, and the inserted nucleotides are denoted with carmine letters. The type of mutations and the numbers of deleted/inserted nucleotides are shown on the right. C, Agrobacterium-mediated expression of Avr4 in N. benthamiana:Cf-4 plants results in a rapid HR at the site of infiltration (upper panel), whereas agro-infiltration of Avr4 failed to induce cell death in the two N. benthamiana:Cf-4 double sobir1/sobir1-like knock-out lines (middle two panels) and in the N. benthamiana:Cf-4 single sobir1 knock-out line (lower panel). D, Complementation by transient expression of NbSOBIR1, SlSOBIR1, or SlSOBIR1-like, restores the Avr4/Cf-4-specific HR in the two N. benthamiana:Cf-4 sobir1/sobir1-like mutants, whereas this complementation does not take place upon transient expression of the corresponding kinase-dead mutants. NbSOBIR1, SlSOBIR1, or SlSOBIR1-like, as well as their corresponding kinase-dead mutants (negative controls), was transiently co-expressed with Avr4, in the leaves of the two N. benthamiana:Cf-4 sobir1/sobir1-like knock-out lines. Each construct was agroinfiltrated at an OD₆₀₀ of 0.5, and the leaves were photographed at 5 dpi. E, Avr4 fails to induce a ROS burst in the two N. benthamiana:Cf-4 sobir1/sobir1-like knock-out lines and in the N. benthamiana:Cf-4 single sobir1 knock-out line. Leaf discs of N. benthamiana:Cf-4 (upper panel), the two N. benthamiana:Cf-4 double sobir1/sobir1-like knock-out lines (middle two panels), and the N. benthamiana:Cf-4 single sobir1 knock-out line (lower panel), were treated with 0.1 μM Avr4 or 0.1 μM flg22 (positive control), or with water (mock) (negative control). ROS production is expressed as relative light units (RLUs), and the data are represented as mean + SD. Experiments were repeated at least three times, and similar results were obtained. Representative pictures are shown. Note that in the N. benthamiana:Cf-4 sobir1/sobir1-like knock-out lines, as well as in the N. benthamiana:Cf-4 single sobir1 knock-out line, the response to flg22 manifests itself as a biphasic ROS burst, whereas in N. benthamiana:Cf-4 the flg22-triggered ROS burst is monophasic.