Studying the cyst nematode resistance gene Hs4 in different genomic backgrounds

My work consisted of four work packages. In the first, I screened a large set of Patellifolia and Beta species for Hs4 homologs. All Patellifolia species contained highly similar Hs4 homologs, while BvHs4 homologs were present in all Beta species analyzed. When examining the expression patterns of thehomologs, I identified substantial differences between species, however, no response to nematode inoculation was observable.

In my second work package, I characterized Arabidopsis T3 lines expressing Hs4 from different promoters. I found lines exhibiting significant nematode resistance compared to the susceptible control under in vitro conditions. The resistance was correlated with the Hs4 expression level.

In the third work package, I analyzed the ultrastructure of syncytia in high-expressing Hs4-transgenic Arabidopsis lines. Syncytia in non-transgenic Arabidopsis were filled with dense cytoplasm, many functional mitochondria, small vacuoles, and numerous smooth ER vesicles. In contrast, in the resistant roots, necrosis of the initial syncytial cell started five days post-inoculation and completely degraded syncytia were detectable three days later. The cytoplasm was filled with large, translucent membranous structures and vacuoles. Most strikingly, I observed spherical ER-derived membrane aggregations with ribosomes on the outer tubules, while smooth ER was almost completely absent. It can be assumed that the rhomboid-protease Hs4, upon attack by the nematode, leads to the membrane aggregation formation.

Rapeseed is a good host for H. schachtii, and the generation of BCN-resistant rapeseed could facilitate crop rotation systems with sugar beet in the future. Therefore, I transformed rapeseed with a 35S::Hs4 construct. I obtained one transgenic plant, and seeds were harvested for future studies.

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