Cloning and characterization of th BPR1 gene ('Botrytis cinerea and Pseudomonas syringae pv. phaseolicola susceptibility and Root length 1') from Arabidopsis thaliana

Analysis of a Pathogen and Circadian Clock controlled Arabidopsis gene, PCC1, revealed five paralogs in the genome of this model plant on chromosomes 1, 2 and 3. The expression pattern of these five members of the PCC1 super-family under various conditions and after particular stimuli was followed by Northern blot analysis with gene-specific oligo probes. One particular paralogue (At1g05340) was found to be associated with susceptibility to pathogens and altered root length and was chosen for further in-depth study. The locus was renamed “BPR1” (Botrytis and Pseudomonas syringae pv. phaseolicola susceptibility and Root length). BPR1 expression was very low in unstressed plants, but was induced by exogenous application of salicylic acid (SA). BPR1 transcripts were detectable by 2 h after infiltration of Arabidopsis with an avirulent strain of Pseudomonas syringae and one day after infiltration with a higher concentration of virulent P. syringae. Transcript levels remained elevated until the second day after treatment. Inoculation with Botrytis cinerea caused accumulation of BPR1 transcripts for 4 consecutive days whereas PCC1 expression decreased. BPR1 and PCC1 expression did not increase after inoculation with Alternaria brassicicola. Pseudomonas-induced BPR1 expression was observed in several defence signalling mutants (eds1, eds5, sid2 and npr1) and the jasmonic acid signalling pathway mutant (coi1). In contrast, Pseudomonas-induced PCC1 expression did not occur in these defence signalling mutants. However, Pseudomonas induced neither BPR1 nor PCC1 expression in NahG plants. Exogenous application of methyljasmonate and ethylene did not induce BPR1 expression by 4 h after treatment. BPR1 induction was dependent on the resistance gene RPS2 and on NDR1. In contrast, PCC1 induction was not dependent on the RPS2 resistance gene. Organ specific expression studies showed BPR1 was expressed in roots, flowers and infected leaves but not in uninfected leaves and stems. By contrast, PCC1 expression was seen only in infected and uninfected leaves. Histochemical localization of GUS activity in transgenic Col-0 plants demonstrated BPR1 expression in vascular tissue of roots, sepals, and petals and in filaments, developing ovules, stigmas, and infected leaves. Over-expression of BPR1 was achieved by use of the constitutive 35S promoter and a knock out BPR1 line was characterized which had a T-DNA insertion in the promoter region of the BPR1 gene. Four-week-old 35S::BPR1 plants exhibited chlorotic symptoms 8 days after P. syringae pv. phaseolicola infiltration. Bacterial re-isolation experiments revealed at least 7 fold more bacteria in over-expression lines than in the wild-type. 35S::BPR1 plants showed increased susceptibility to Botrytis cinerea in comparison to wild-type and knockout BPR1 plants. In a symptom category from 1 (low) to 5 (intensive), most over-expression plants were placed to categories 4 and 5, in contrast the majority of wild-type and knockout plants which were placed into categories 2 and 3.Roots of 35S::BPR1 plants were almost 30% longer than knockout BPR1. The data from the root length measurements were analyzed by completely randomized design which came from 2 over-expression lines, knockout and wild-type plants. The means of the root lengths were compared by Duncan’s test and differences were found to be significant at the P<1% level, BPR1 knock-out, wild-type and over expression plants comprised 3 separate classes: a, b and c from the shortest to longest roots, respectively. To study genetic polymorphisms in the PCC1 superfamily we chose 10 accessions (Ct-1, Oy-0, Bur-0, Cvi-0, St-0, Shakdara, Mt-0, Mh-1, Ms-0, Ta-0) of Arabidopsis for which McKhann et al. (2004) have shown in genetic differences in four genes. Member-specific primers were chosen from the 5`UTR and 3`UTR regions. Polymorphisms were seen for all PCC1 superfamily members. A PCC1 natural mutant was found in some accessions which showed a 145 bp smaller PCR band. Sequence analysis revealed a deletion of the end of exon 2, of the entire intron 2 and the beginning and the end of exon 3. Additionally there were 4 single nucleotide substitutions in exon 2 and exon 3. Rhythmic transcript fluctuations of the PCC1 natural mutant suggest normal expression at least at the RNA level. BPR1 protein was expressed as a GST (Gluthation S transferase) fusion in E. coli to produce polyclonal antibody. To study BPR1 protein interactions, the lexA yeast-two-hybrid system was used. The BPR1-bait vector was tested in yeast for protein expression and self-activation. A lexA yeast two hybrid cDNA library from pathogen-challenged Arabidopsis was amplified. A preliminary experiment indicated that BPR1 protein might interact with At2g39010, an aquaporin that is known as an intrinsic plasma membrane protein with water channel activity, and water transport protein. In summary, BPR1 and PCC1 appear to have a rather complementary instead of a redundant expression pattern. From my results, BPR1 appears to be a susceptibility factor for certain pathogens in Arabidopsis.

Grantor dc:publisher

Publikationsserver der RWTH Aachen University

Year dc:date

2006

Author dc:creator

• Sharifi-Sirchi, Gholam Reza

Contributors dc:contributor

• Slusarenko, Alan

• info:eu-repo/classification/ddc/570
• Biowissenschaften, Biologie

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

eng

Identifier

https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-122757%22

OAI identifier oai:identifier

oai:publications.rwth-aachen.de:61073