Fusarium verticillioides (Fv) is a major cereal pathogen causing stalk rot and ear rot in maize, negatively affecting crop productivity, and compromising food safety by producing the secondary metabolites fumonisins. Several studies were conducted to identify maize genes associated with host plant resistance to Fv infection and fumonisin accumulation. The maize WRKY transcription factors and the lipoxygenases (ZmLOXs) are well recognized as important players in plant defense against pathogens, and it is known that the host-pathogen lipid cross-talk influences the pathogenesis. In this regard, previous RNA-seq experiments reported the enhanced expression of ZmLOX genes in maize resistant genotypes and GWAS resulted in one SNP significantly associated with ZmWRKY125. The Clustered Regularly Interspaced Short Palindromic Repeat/associated Cas9 (CRISPR/Cas9) editing of ZmWRKY125 and the transgenic overexpression of ZmLOX4 genes were carried out to investigate the possible implication of these two genes in the resistance mechanisms against Fv. Before cloning experiments, protein domain conservation and different splicing products have been analyzed comparing homologues and orthologues for both genes. As regards ZmWRKY125, the CRISPR cloning was based on a double cloning using two different guides (sgRNA) for one gene target. Agrobacterium tumefaciens mediated transformation was used for introducing the construct under the maize promoter ZmpUBI in the binary vector p1609 in maize A188 line. Mutants from three different transformation events were obtained. For each event, T2 plants will be genotyped to find homozygous for the mutation that in turn will be phenotyped for Fv resistance and fumonisin content. As regards ZmLOX4, the gene was cloned under an overexpressed promoter involved in kernel development in the vector L1781, and the same transformation conditions adopted for the CRISPR/Cas9 editing of ZmWRKY125 were used. Mutants from two different transformation events were obtained. For each event, T2 plants were genotyped in order to find homozygous for the mutation. Homozygous plants will be further evaluated for Fv resistance, fumonisin accumulation, oxylipin content as well as for the expression analysis of the main genes involved in the jasmonic acid pathway.

Ottaviani, L., Rogowsky, P., Marocco, A., Lanubile, A., THE CRISPR/CAS9 EDITING OF A WRKY GENE AND THE OVEREXPRESSION OFA LIPOXYGENASE GENE FOR IMPROVING PATHOGEN RESISTANCE IN MAIZE, Abstract de <<LXV SIGA Annual Congress>>, (Piacenza, 06-09 September 2022 ), Società Italiana di Genetica Agraria, Piacenza 2022: 1-2 [http://hdl.handle.net/10807/217705]

THE CRISPR/CAS9 EDITING OF A WRKY GENE AND THE OVEREXPRESSION OF A LIPOXYGENASE GENE FOR IMPROVING PATHOGEN RESISTANCE IN MAIZE

Ottaviani, L.;Marocco, A.;Lanubile, A.
2022

Abstract

Fusarium verticillioides (Fv) is a major cereal pathogen causing stalk rot and ear rot in maize, negatively affecting crop productivity, and compromising food safety by producing the secondary metabolites fumonisins. Several studies were conducted to identify maize genes associated with host plant resistance to Fv infection and fumonisin accumulation. The maize WRKY transcription factors and the lipoxygenases (ZmLOXs) are well recognized as important players in plant defense against pathogens, and it is known that the host-pathogen lipid cross-talk influences the pathogenesis. In this regard, previous RNA-seq experiments reported the enhanced expression of ZmLOX genes in maize resistant genotypes and GWAS resulted in one SNP significantly associated with ZmWRKY125. The Clustered Regularly Interspaced Short Palindromic Repeat/associated Cas9 (CRISPR/Cas9) editing of ZmWRKY125 and the transgenic overexpression of ZmLOX4 genes were carried out to investigate the possible implication of these two genes in the resistance mechanisms against Fv. Before cloning experiments, protein domain conservation and different splicing products have been analyzed comparing homologues and orthologues for both genes. As regards ZmWRKY125, the CRISPR cloning was based on a double cloning using two different guides (sgRNA) for one gene target. Agrobacterium tumefaciens mediated transformation was used for introducing the construct under the maize promoter ZmpUBI in the binary vector p1609 in maize A188 line. Mutants from three different transformation events were obtained. For each event, T2 plants will be genotyped to find homozygous for the mutation that in turn will be phenotyped for Fv resistance and fumonisin content. As regards ZmLOX4, the gene was cloned under an overexpressed promoter involved in kernel development in the vector L1781, and the same transformation conditions adopted for the CRISPR/Cas9 editing of ZmWRKY125 were used. Mutants from two different transformation events were obtained. For each event, T2 plants were genotyped in order to find homozygous for the mutation. Homozygous plants will be further evaluated for Fv resistance, fumonisin accumulation, oxylipin content as well as for the expression analysis of the main genes involved in the jasmonic acid pathway.
Inglese
Proceedings of the LXV SIGA Annual Congress
LXV SIGA Annual Congress
Piacenza
6-set-2022
9-set-2022
978-88-944843-3-5
Società Italiana di Genetica Agraria
Ottaviani, L., Rogowsky, P., Marocco, A., Lanubile, A., THE CRISPR/CAS9 EDITING OF A WRKY GENE AND THE OVEREXPRESSION OFA LIPOXYGENASE GENE FOR IMPROVING PATHOGEN RESISTANCE IN MAIZE, Abstract de <<LXV SIGA Annual Congress>>, (Piacenza, 06-09 September 2022 ), Società Italiana di Genetica Agraria, Piacenza 2022: 1-2 [http://hdl.handle.net/10807/217705]
File in questo prodotto:
File Dimensione Formato  
The_CRISPR_CAS9_editing_of_a_WRKY_gene_and_the_overexpression_of_a_lipoxygenase_gene_for_improving_pathogen_resistance_in_maize.pdf

accesso aperto

Licenza: Non specificato
Dimensione 665.54 kB
Formato Adobe PDF
665.54 kB Adobe PDF Visualizza/Apri

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10807/217705
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus ND
  • ???jsp.display-item.citation.isi??? ND
social impact