The vectors construction
To construct vectors for overexpression, the full-length ORF ofCaNAC2c was cloned into the entry vector pDONR207 by BP reaction
with appropriate primers (Table S1) and then cloned into destination
vectors pEarleyGate103, pDEST-15 and PK7WG2 by LR reaction, using
Gateway cloning techniques (Invitrogen, Carlsbad, CA, USA). Two specific
300-400 bp fragments in the ORF and 3’UTRs of CaNAC2c /dand CaHSFA5, which were confirmed by BLAST searching against
genome sequence in the databases of Zunla-1
(http://peppersequence.genomics.cn/page/species/blast.jsp), were used
for vector construction for CaNAC2c /d orCaHSFA5 VIGS silencing. The specific fragments were cloned into
the entry vector pDONR207 and then into the PYL279 vector. To construct
vectors for 5’ series deletions assay of pCaNAC2c, the full
length pCaNAC2c and its 5’ series deletions were first cloned
into pDONR207 by BP reaction with appropriate primers (Table S2) and the
into the pMDC163 destination vector by LR reaction using Gateway cloning
techniques for assaying expression of β-glucuronidase (GUS) driven bypCaNAC2c or its 5’series deletions in leaves of pepper plants by
transient overexpression through agro-infiltration.