This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
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Zymoseptoria tritici is a fungal plant pathogen affecting wheat crops around the globe causing the disease known as ‘septoria leaf blotch’, resulting in necrotic brown blotches on the wheat’s foliage. Z. tritici has been particularly problematic to control. It secretes the LysM domain effector proteins Zt3LysM, Zt1LysM and Ztx1LysM which essentially ‘mop up’, or sequester, any chitin shed by the pathogen. Since chitin is usually detected by the wheat, triggering defences, these effectors enable Z. tritici to go undetected by the plant. It also protects Z. tritici against host chitinases. Of these proteins, Zt3LysM is essential for full virulence on the host, with gene disruption strains showing severely reduced colonisation and reproduction.
This project focuses on whether proteins with sequence homology to Zt3LysM would ‘complement’ the function of Zt3LysM and restore virulence in a Zt3LysM knockout strain. To do this, proteins with sequence homology to Zt3LysM were identified using a BLASTP search on JGI Mycocosm. Proteins were selected from species with a range of lifestyles, from phytopathogens to arbuscular mycorrhizal fungi to arthropod parasites. The species selected were Cladosporium fulvum (tomato pathogen with a known orthologue), Gaeumannomyces graminis var. tritici (cereal root pathogen), Botrytis cinerea (necrotrophic phytopathogen), Lentinula boryana (Saprophyte – white rot), Trichophyton mentagrophytes (causes ringworm), Rhizophagus irregularis (arbuscular mycorrhizal fungus), and Beauveria bassiana (arthropod parasite).
In order to generate complementation strains, this project focused on generating Gibson assembled plasmids containing the Zt3LysM promoter, CDS of each gene of interest, and Zt3LysM terminator region. These will be used for transformation into Zt3LysM deletion strains.
To start off with, PCR reactions and gel electrophoresis tests were conducted to validate that the primers were effective. Next, PCRs of all the required fragments were made which were subsequently purified and gel extracted to collect the concentrated DNA in preparation for Gibson assembly. For each gene of interest, including Zt3LysM as the control, the Zt3LysM promoter, terminator, and the CDS were incorporated together with a pcGEN vector backbone. These constructs were then used to transform E. coli, and positive transformants were selected using kanamycin antibiotic. Thereafter, a colony PCR was performed to verify which colonies of E. coli contained the correctly aligned reconstructed plasmid. Those colonies which appeared to have taken up the plasmid were chosen for the miniprep to isolate the cloned plasmids from the E. coli. Further checks in the form of restriction digests were then performed.
In summary, the final product for all candidate genes after being isolated from the E. coli should contain the pcGEN backbone, the Zt3LysM promoter and terminator, and the coding sequence of the orthologue gene. This was the extent of my 10 week internship working at Rothamsted Research, however there are plans to continue the project by using these constructs to transform Agrobacterium, then performing Agrobacterium-mediated transformation of Z. tritici Zt3LysM knockout strains. These will then be swabbed onto wheat leaves to determine whether the Zt3LysM-dependent full virulence has been restored, or complemented, by any of the candidates.
I very much enjoyed working at Rothamsted Research and have learned several new lab techniques which will help me in my degree and any future projects I’ll take part in. In addition, after being taught the methods of the project, I was often working independently in the lab which helped me improve my self-discipline and time management skills. I would like to thank my supervisor Luca Steel for all the support and help on the project, as well as the BSPP for this opportunity.