Plasmid construction and rice transformation
To evaluate the PPik-H4 activity, the promoter region of Pik-H4 was cloned from Pik-H4 NILs leaves with CTAB extraction and was inserted into a binary vector pGRRFP ::GFP . Then, the pGR vector was introduced into Nipponbare via Agrobacterium -mediated (EHA105) transformation. For promoter split and mutation assay, series plasmids were constructed with multi-fragment homologous recombination PCR. For GUS staining, PPik1-H4 and PPik2-H4 were cloned to pCAMBIA1305.1 and induced into Nipponbare. The coding sequences of Pik1-H4 andPik2-H4 were inserted into the pOX-GFPPUbi ::GFP vector and transformed into LTH orPik-H4 NILs. Pik1-H4 ,Pik2-H4 and AvrPik-E coding sequences were cloned to pEAQ(Sainsbury, Thuenemann and Lomonossoff, 2009) with reconstruction: pEAQ-FlagP35S ::Flag , pEAQ-HAP35S ::HA and pEAQ-MycP35S ::Myc respectively for tobacco transient expression.