Construction of a tomato leaf cDNA expression library and immunoscreening for the Pseudomonas syringae pv. tomato DC3000 HrpZ1 target protein

Pseudomonas syringae pv. tomato DC3000 is a Gram-negative plant pathogen that causes bacterial speck disease on tomato. The virulence of this bacterium is based on the type III secretion system (T3SS). Similar systems are also used by many other plant and animal pathogens, as well as symbiotic bacteria. The T3SS enables the transfer of specific bacterial virulence proteins from the bacterial cytoplasm into the host cell. This secretion is mediated by a needle-like structure that penetrates the plant cell wall. Once inside the host cells, the effector proteins are capable of shutting down the host’s immune system. However, what happens at the plant cell membrane is not well understood. One of the first bacterial proteins that come into interaction with a host protein during P. syringae pv. tomato DC3000 infection is the HrpZ1 protein that is believed to participate in a membrane interaction, facilitating the transfer of effector proteins. Previous research has shown that HrpZ1 binds to a peptide and using an antiserum raised against this peptide in immunoblotting tests of tomato proteins separated by SDS-PAGE and isoelectric focusing, the target tomato protein of HrpZ1 has been found to be small and acidic. However, this specific protein has not yet been fully characterized. This is why the goal of the work for this thesis was to identify and characterize the target protein of HrpZ1 in tomato. For this purpose, a lambda cDNA expression library from tomato leaves was constructed, followed by immunoscreening of the library with the abovementioned antiserum, and analysing candidate clones by sequencing. Even though a good cDNA library was obtained, the immunoscreenings did not yield satisfactory results. Thus, the pursuit of the HrpZ1 target protein needs to be continued. %%%%Pseudomonas syringae pv. tomato DC3000 ar en Gram-negativ vaxtpatogen som orsakar sjukdomen bacterial speck hos tomat. Bakteriens virulens baserar sig pa ett system kallat typ III sekretion (T3SS). Liknande system anvands ocksa av manga andra vaxt- och djurpatogener, saval som av symbiotiska bakterier. Detta system mojliggor overforing av specifika bakterieproteiner som medverkar i virulensen fran bakteriens cytoplasma till vardcellen. Sekretionen sker med hjalp av en struktur liknande en injektionsnal, som tranger igenom vaxtens cellvagg och plasmamembran. Inne i vardcellen kan dessa sa kallade effektorproteiner rasera vardens immunsystem. Ett av de forsta bakterieproteinerna som kommer i kontakt med ett vardprotein vid en infektion av P. syringae pv. tomato DC3000, ar proteinet HrpZ1 vilket tros delta i en membraninteraktion och forebygga transporten av effektorproteiner. Tidigare forskning har visat att HrpZ1 binder till en viss peptid, och genom anvandning av ett antiserum mot denna peptid i immunoblot-tester med tomatproteiner som separerats med SDS-PAGE och isoelektrisk fokusering, har man kunnat konstatera att tomatproteinet som HrpZ1 binder till ar litet och surt. Proteinet som HrpZ1 interagerar med har dock inte annu helt karakteriserats. …