CRISPR/Cas9-induced knockout of an amino acid permease gene (AAP6) reduced Arabidopsis thaliana susceptibility to Meloidogyne incognita

Abstract

Background: Plant-parasitic root-knot nematode (Meloidogyne incognita) causes global yield loss in agri- and horticultural crops. Nematode management options rely on chemical method. However, only a handful of nematicides are commercially available. Resistance breeding efforts are not sustainable because R gene sources are limited and nematodes have developed resistance-breaking populations against the commercially available Mi-1.2 gene-expressing tomatoes. RNAi crops that manage nematode infection are yet to be commercialized because of the regulatory hurdles associated with transgenic crops. The deployment of the CRISPR/Cas9 system to improve nematode tolerance (by knocking out the susceptibility factors) in plants has emerged as a feasible alternative lately.

Results: In the present study, a M. incognita-responsive susceptibility (S) gene, amino acid permease (AAP6), was characterized from the model plant Arabidodpsis thaliana by generating the AtAAP6 overexpression line, followed by performing the GUS reporter assay by fusing the promoter of AtAAP6 with the β-glucuronidase (GUS) gene. Upon challenge inoculation with M. incognita, overexpression lines supported greater nematode multiplication, and AtAAP6 expression was inducible to the early stage of nematode infection. Next, using CRISPR/Cas9, AtAAP6 was selectively knocked out without incurring any growth penalty in the host plant. The 'Cas9-free' homozygous T₃ line was challenge inoculated with M. incognita, and CRISPR-edited A. thaliana plants exhibited considerably reduced susceptibility to nematode infection compared to the non-edited plants. Additionally, host defense response genes were unaltered between edited and non-edited plants, implicating the direct role of AtAAP6 towards nematode susceptibility.

Conclusion: The present findings enrich the existing literature on CRISPR/Cas9 research in plant-nematode interactions, which is quite limited currently while compared with the other plant-pathogen interaction systems.

Keywords: R gene; S gene; Amino acid transporter; Gene expression; Multiplication ratio; Mutation.

Fig. 1

AAP6 is greatly conserved in dicotyledonous plants and ubiquitously expressed in A. thaliana. (a) Evolutionary relationships of the AtAAP6 protein (entry is indicated in bold font) with its corresponding orthologues from other plant families. The phylogenetic tree was constructed using the ML method. Bootstrap consensus was inferred from 1000 replicates, and branches are supported by > 70% of replicates. The NCBI accession numbers of 52 different entries are provided in parentheses. The tree was rooted with the O. sativa subsp. japonica AAP6 protein as the out-group. Entries in different colors represent different plant families, as written aside the specific clusters. Solid rectangles in different colors indicate different plant orders. (b) The comparative amino acid sequence identity of AAP6 with its paralogues (AAP1, AAP2, AAP3, AAP4, AAP5, AAP7, and AAP8) from A. thaliana. (c, d) RT-qPCR-based expression analysis of the AAP6 gene in different plant parts and developmental stages of A. thaliana ecotype Columbia-0. Fold change in expression was set at 1 in root tissue and 7 days-old-plant, and statistically compared with AAP6 expression in other plant parts and developmental stages, respectively (no significant difference was observed; Tukey’s HSD test, P > 0.01). Gene expression was normalized using two housekeeping genes of A. thaliana (ubiquitin and 18 S rRNA). Each bar represents the mean fold change value ± standard error (SE) of qPCR runs in five biological and three technical replicates

Fig. 2

AtAAP6 overexpression increased A. thaliana susceptibility to M. incognita. (a) Schematic representation of the T-DNA regions corresponding to the AtAAP6 overexpression vector (driven by the CaMV35S promoter) and promoter::GUS fusion vector (gusA expression is driven by the promoter of the AtAAP6 gene). T_NOS, T_35S – polyadenylation signals of nopaline synthase and CaMV35S for transcription termination. Arrows indicate the direction of transcription. Hygromycin (Hyg) was used as the selectable marker. LB, RB – left and right borders. (b) RT-qPCR-based detection of an increase in AtAAP6 mRNA abundance in an overexpression line at 3, 10, 15 and 20 days post inoculation (dpi) of M. incognita. Asterisks (*P < 0.01, **P < 0.0001; paired t-test) represent significant differential expression of the AtAAP6 transcript in nematode-infected wild-type and overexpression line when compared to the baseline expression in uninfected control plants (fold change values were set at 1). Gene expression data was normalized using two reference genes, i.e., A. thaliana 18 S rRNA and ubiquitin. Bars represent the mean fold change value of five biological and three technical replicates ± standard errors. (c) Numbers of gall, female, egg per egg mass, and MF ratio per root system were significantly higher in the overexpression line than the wild-type at 30 dpi. Bars represent the mean of five replications ± standard errors. Asterisks indicate a significant difference between two treatments (*P < 0.05, **P < 0.01; two-way ANOVA followed by Tukey’s significant difference test). (d) RT-qPCR-based validation of gusA gene expression in a transformed line (harboring the promoter::GUS fusion) at 0, 3, 7, 10, 15, 20 and 25 dpi. The fold change in gene expression was set at 1 in the uninfected control and compared with other treatments. Other parameters were kept as identical as described above. Bars with different letters are significantly different at P < 0.01, one-way ANOVA followed by Tukey’s test

Fig. 3

Strong and localized expression of AtAAP6 in the galled root of A. thaliana upon M. incognita infection. Expression of pAAP6::GUS in the growing tissues of a three-week-old plant: (a) shoot, (b) leaf, (c) root. Expression of pAAP6:GUS in nematode-infected root segments at different days post inoculation (dpi): (d) 0 dpi, (e) 3 dpi, (f) 7 dpi, (g) 10 dpi, (h) 15 dpi, (i) 20 dpi Scale bar = 100 μm

Fig. 4

Targeted knockout of the AtAAP6 gene in A. thaliana using the CRISPR/Cas9 system. (a) Two gRNA spacer sequences (target 1 and 2 are in the negative and positive strand, respectively) were designed from exon 2 of the AtAAP6 gene. Protospacer adjacent motif (PAM) sites are bold and italicized. (b) The T-DNA portion of the recombinant Cas9-expressing vector (pHEE401:AtAAP6) is schematically illustrated. Two gRNA expression cassettes (guide RNA + scaffold) were assembled via Golden Gate cloning. gRNA and Cas9 expression were driven by the Arabidopsis U6 promoter and an egg cell-specific promoter (EC1p), respectively. NLS – nuclear localization signal, 35Sp – CaMV35S promoter, HygR – Hygromycin resistance, LB, RB – left and right borders. (c) Sequencing-based identification of edited events in the T₀ generation of plants. Four different types of mutations, i.e., homozygous, heterozygous, bi-allelic, and chimeric were detected with a maximum deletion (red hyphens) and insertion (green letter) of 6 and 1 bp, respectively. (d) Summarized data shows the editing efficiency and specific number of different mutant genotypes obtained. (e) The predicted mutated proteins in different events are schematically represented. AtAAP6 was heavily truncated (due to premature translation termination) in a homozygous event, AtAAP6-cr-5. TM – transmembrane domains

Fig. 5

Targeted mutagenesis of AtAAP6 conferred improved resistance in A. thaliana against M. incognita infection. (a) Growth phenotypes of wild-type (WT) and mutant plants at two weeks after germination in MS agar. Scale bar = 3 cm. (b) Shoot morphology of WT and mutant plants at 30 days after germination in soil. (c) Photomicrographs depict lower galling intensity in a mutant root system compared to the WT root system at 30 days post inoculation (dpi) of nematodes. Magnified images at the bottom show the developmental delay of nematodes in mutant roots because WT roots harbored females (F), whereas mutants harbored J3/J4 spike-tail stages at that time. Asterisks indicate the location of putative feeding cells. Scale bar = 500 μm. (d) Numbers of gall, female, egg per egg mass, and MF ratio per root system were reduced in mutants compared to WT at 30 dpi. Bars represent the mean of five replications ± standard error. Different letters indicate a significant difference (within a specific infection parameter) at P < 0.0001, a two-way ANOVA followed by Tukey’s significant difference test