A simplified procedure for the subtractive cDNA cloning of photoassimilate-responding genes: isolation of cDNAs encoding a new class of pathogenesis-related proteins.
Transgenic tobacco plants (ppa-1) constitutively expressing Escherichia coli pyrophosphatase behind the 35S CaMV promoter accumulate high levels of soluble sugars in their leaves [27]. These plants were considered a tool to study adaptation of leaves to photoassimilate accumulation at the molecular level. By differential hybridization of a subtractive library enriched for transcripts present in the transgenic plants 12 different cDNAs were isolated. By sequence analysis four cDNAs could be identified as 1-aminocyclopropane-1-carboxylate-oxidase and as three different pathogenesis-related proteins (PR-1b, PR-Q and SAR 8.2). Two cDNAs were homologous to a calmodulin-like protein from Arabidopsis and a human ribosomal protein L19 while six cDNA clones remained unknown. One of these clones (termed PAR-1 for photoassimilate-responsive) displayed features similar to pathogenesis-related proteins: Hybridizing transcripts, 1.2 and 1.0 kb in length, were strongly inducible by salicylate and accumulated in tobacco plants after infection with potato virus Y (PVY) both in infected and uninfected systemic leaves. PAR-1 transcripts also accumulated in wildtype leaves upon floating on glucose and sucrose whereas sorbitol and polyethylene glycol had no effect. Rescreening of the ppa-1 cDNA library with the PAR-1 cDNA as probe resulted in 25 hybridizing cDNAs which by homology were found to fall into three classes (PAR-1a, b, c). The cDNAs coding for PAR-1a and b were 90.6% homologous on the DNA level while both were less related to the PAR-1c cDNA (70.5% and 75.2% homologous, respectively). One open reading frame was identified in all three PAR-1 cDNA classes. Translation would result in proteins with a theoretical molecular mass of about 20 kDa. The N-terminal amino acid sequences resemble a signal peptide which would direct the proteins to the secretory pathway. Using selective 3' hybridization probes of the three PAR-1 cDNAs it was possible to discriminate the different transcripts. Both PAR-1a and PAR-1c mRNAs are induced in plants treated with PVY.
Herbers
K
K
Institut für Pflanzengenetik und Kulturpflanzenforschung, Gatersleben, Germany.
Mönke
G
G
Badur
R
R
Sonnewald
U
U
D000595:Amino Acid Sequence; D017796:Caulimovirus / Q000235:genetics; D003001:Cloning, Molecular; D018076:DNA, Complementary; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D005947:Glucose / Q000494:pharmacology; D008027:Light; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics*; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D017800:Potyvirus / Q000502:physiology; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D012459:Salicylates / Q000494:pharmacology; D020156:Salicylic Acid; D017386:Sequence Homology, Amino Acid; D013395:Sucrose / Q000494:pharmacology; D014026:Nicotiana / Q000235:genetics / Q000502:physiology / Q000821:virology
D018076:DNA, Complementary; D010940:Plant Proteins; D012333:RNA, Messenger; D012459:Salicylates; C053376:pathogenesis-related proteins, plant; D013395:Sucrose; D005947:Glucose; D020156:Salicylic Acid