Mutations in Two Aphid-Regulated β-1,3-Glucanase Genes by CRISPR/Cas9 Do Not Increase Barley Resistance to Rhopalosiphum padi L

Abstract

Callose deposition is induced in plants by various stress factors such as when plants are attacked by herbivores and pathogens. In the case of aphids, callose plugging of aphid-damaged phloem sieve tubes is expected to reduce aphid access to the phloem sap, while aphid-induced upregulation of callose-degrading β -1,3-glucanase genes in the host plant might counteract this negative effect on aphid performance. We have tested this hypothesis with barley mutants in which one or both of two β -1,3-glucanase genes (1636 and 1639) have been mutated by CRISPR/Cas9 technique in cv. Golden Promise. These two genes were previously found to be upregulated by the cereal pest Rhopalosiphum padi L. in susceptible barley genotypes. Four 1636/1639 double mutant, three 1636 single mutant and two 1639 single mutant lines were tested for aphid resistance along with control lines. All mutant lines had single base insertions, causing frame shifts and premature stop codons. Three of the four double mutant lines showed significantly reduced β -1,3-glucanase activity, and bacterial flagellin-induction resulted in significantly more callose formation in the leaves of double mutant compared to control and single mutant lines. However, we found no effect of these modified plant traits on barley resistance to R. padi . Both genes were confirmed to be upregulated by R. padi in Golden Promise. The gene 1637 is another β -1,3-glucanase gene known to be upregulated by R. padi in barley and was here found to be higher expressed in a double mutant line when compared with a control line. If this can compensate for the general reduction of β -1,3-glucanase activity in the double mutants is difficult to discern since phloem concentrations of these proteins are unknown.

Efficient Protoplast Regeneration Protocol and CRISPR/Cas9-Mediated Editing of Glucosinolate Transporter (GTR) Genes in Rapeseed (Brassica napus L.)

“…Genomic and full-length open reading frames of six BnGTR1 and six BnGTR2 paralogs were amplified from genomic DNA and cDNA of cv. Kumily, respectively, using gene specific primers according to published protocols (Kim et al, 2020 ; Muthusamy et al, 2020 ), with minor modifications, and confirmed by sequencing. Genomic DNA sequences of different paralogs from the BnGTR1 and BnGTR2 were aligned to find conserved target sites among the paralogs of each gene.…”

Genome editing of oilseed species by CRISPR/Cas9 for trait improvement

“…Genomic and full-length open reading frames of six BnGTR1 and six BnGTR2 paralogs were amplified from genomic DNA and cDNA of cv. Kumily, respectively, using gene specific primers according to published protocols (Kim et al, 2020;Muthusamy et al, 2020), with minor modifications, and confirmed by sequencing. Genomic DNA sequences of different paralogs from the BnGTR1 and BnGTR2 were aligned to find conserved target sites among the paralogs of each gene.…”

Genome editing in cereal crops: an overview

“…Barley mutants with knocked out hvmorc1 further showed de-repression of transposable elements which could not be seen in the RNAi lines but was observed in the Arabidopsis mutants, demonstrating that different strategies to create mutants may result in distinct outcomes. Another study addressing pest resistance showed that combined knock-out of two of three aphid-induced beta-1,3-glucanase genes led to increased callose formation in leaves, however the effect did not lead to enhanced aphid resistance (Kim et al 2020).…”

“…Multiplexing approaches in barley have been carried out to increase the chance for disrupting a given gene or for the generation of fragment deletions at a particular locus (Kapusi et al 2017;Kapusi and Stöger 2018;Li et al 2020a;Zeng et al 2020a), or for the generation of double mutants at two loci (Gasparis et al 2018;Kim et al 2020). Different strategies were used to deliver the gRNAs: a polycistronic tRNA-sgRNA cassette (Gasparis et al 2018;Zeng et al 2020a), two conventional sgRNA cassettes on one vector (Kim et al 2020;Li et al 2020a) and mixing agrobacterium strains transformed with a vector containing each a single sgRNA cassette or, in the case of particle bombardment, co-bombardment of the two vectors (Kapusi et al 2017;Kapusi and Stöger 2018). The probability of isolating individuals edited at two or more target sequences results from the combined efficiencies of the sgRNAs.…”

“…The probability of isolating individuals edited at two or more target sequences results from the combined efficiencies of the sgRNAs. Reported efficiencies for two gRNAs (number of double mutant T0 individuals per total T0 mutant individuals) are for example 4,7% (Kim et al 2020) (Zhang et al 2017). II Multiplexing with two sgRNAs, each targeting two different sites in six homeologs (Brauer et al 2020).…”