Comparative Analysis of Type III Secreted Effector Genes Reflects Divergence of Acidovorax citrulli Strains into Three Distinct Lineages.

Acidovorax citrulli causes bacterial fruit blotch of cucurbits, a serious economic threat to watermelon (Citrullus lanatus) and melon (Cucumis melo) production worldwide. Based on genetic and biochemical traits, A. citrulli strains have been divided into two distinct groups: group I strains have been mainly isolated from various non-watermelon hosts, while group II strains have been generally isolated from and are highly virulent on watermelon. The pathogen depends on a functional type III secretion system for pathogenicity. Annotation of the genome of the group II strain AAC00-1 revealed 11 genes encoding putative type III secreted (T3S) effectors. Due to the crucial role of type III secretion for A. citrulli pathogenicity, we hypothesized that group I and II strains differ in their T3S effector repertoire. Comparative analysis of the 11 effector genes from a collection of 22 A. citrulli strains confirmed this hypothesis. Moreover, this analysis led to the identification of a third A. citrulli group, which was supported by DNA:DNA hybridization, DNA fingerprinting, multilocus sequence analysis of conserved genes, and virulence assays. The effector genes assessed in this study are homologous to effectors from other plant-pathogenic bacteria, mainly belonging to Xanthomonas spp. and Ralstonia solanacearum. Analyses of the effective number of codons and gas chromatography content of effector genes relative to a representative set of housekeeping genes support the idea that these effector genes were acquired by lateral gene transfer. Further investigation is required to identify new T3S effectors of A. citrulli and to determine their contribution to virulence and host preferential association.

Sustaining yield and nutritional quality of peanuts in harsh environments: Physiological and molecular basis of drought and heat stress tolerance.

Climate change is significantly impacting agricultural production worldwide. Peanuts provide food and nutritional security to millions of people across the globe because of its high nutritive values. Drought and heat stress alone or in combination cause substantial yield losses to peanut production. The stress, in addition, adversely impact nutritional quality. Peanuts exposed to drought stress at reproductive stage are prone to aflatoxin contamination, which imposes a restriction on use of peanuts as health food and also adversely impact peanut trade. A comprehensive understanding of the impact of drought and heat stress at physiological and molecular levels may accelerate the development of stress tolerant productive peanut cultivars adapted to a given production system. Significant progress has been achieved towards the characterization of germplasm for drought and heat stress tolerance, unlocking the physiological and molecular basis of stress tolerance, identifying significant marker-trait associations as well major QTLs and candidate genes associated with drought tolerance, which after validation may be deployed to initiate marker-assisted breeding for abiotic stress adaptation in peanut. The proof of concept about the use of transgenic technology to add value to peanuts has been demonstrated. Advances in phenomics and artificial intelligence to accelerate the timely and cost-effective collection of phenotyping data in large germplasm/breeding populations have also been discussed. Greater focus is needed to accelerate research on heat stress tolerance in peanut. A suits of technological innovations are now available in the breeders toolbox to enhance productivity and nutritional quality of peanuts in harsh environments. A holistic breeding approach that considers drought and heat-tolerant traits to simultaneously address both stresses could be a successful strategy to produce climate-resilient peanut genotypes with improved nutritional quality.

Base Editing in Peanut Using CRISPR/nCas9.

Peanut (Arachis hypogaea L.), an allotetraploid legume of the Fabaceae family, is able to thrive in tropical and subtropical regions and is considered as a promising oil seed crop worldwide. Increasing the content of oleic acid has become one of the major goals in peanut breeding because of health benefits such as reduced blood cholesterol level, antioxidant properties and industrial benefits such as longer shelf life. Genomic sequencing of peanut has provided evidence of homeologous AhFAD2A and AhFAD2B genes encoding Fatty Acid Desaturase2 (FAD2), which are responsible for catalyzing the conversion of monounsaturated oleic acid into polyunsaturated linoleic acid. Research studies demonstrate that mutations resulting in a frameshift or stop codon in an FAD2 gene leads to higher oleic acid content in oil. In this study, two expression vectors, pDW3873 and pDW3876, were constructed using Cas9 fused to different deaminases, which were tested as tools to induce point mutations in the promoter and the coding sequences of peanut AhFAD2 genes. Both constructs harbor the single nuclease null variant, nCas9 D10A, to which the PmCDA1 cytosine deaminase was fused to the C-terminal (pDW3873) while rAPOBEC1 deaminase and an uracil glycosylase inhibitor (UGI) were fused to the N-terminal and the C-terminal respectively (pDW3876). Three gRNAs were cloned independently into both constructs and the functionality and efficiency were tested at three target sites in the AhFAD2 genes. Both constructs displayed base editing activity in which cytosine was replaced by thymine or other bases in the targeted editing window. pDW3873 showed higher efficiency compared to pDW3876 suggesting that the former is a better base editor in peanut. This is an important step forward considering introgression of existing mutations into elite varieties can take up to 15 years making this tool a benefit for peanut breeders, farmers, industry and ultimately for consumers.

CRISPR/Cas9 Based Site-Specific Modification of FAD2 cis-Regulatory Motifs in Peanut (Arachis hypogaea L).

Peanut (Arachis hypogaea L.) seed is a rich source of edible oil, comprised primarily of monounsaturated oleic acid and polyunsaturated linoleic acid, accounting for 80% of its fatty acid repertoire. The conversion of oleic acid to linoleic acid, catalyzed by Fatty Acid Desaturase 2 (FAD2) enzymes, is an important regulatory point linked to improved abiotic stress responses while the ratio of these components is a significant determinant of commercial oil quality. Specifically, oleic acid has better oxidative stability leading to longer shelf life and better taste qualities while also providing nutritional based health benefits. Naturally occurring FAD2 gene knockouts that lead to high oleic acid levels improve oil quality at the potential expense of plant health though. We undertook a CRISPR/Cas9 based site-specific genome modification approach designed to downregulate the expression of two homeologous FAD2 genes in seed while maintaining regulation in other plant tissues. Two cis-regulatory elements the RY repeat motif and 2S seed protein motif in the 5'UTR and associated intron of FAD2 genes are potentially important for regulating seed-specific gene expression. Using hairy root and stable germ line transformation, differential editing efficiencies were observed at both CREs when targeted by single gRNAs using two different gRNA scaffolds. The editing efficiencies also differed when two gRNAs were expressed simultaneously. Additionally, stably transformed seed exhibited an increase in oleic acid levels relative to wild type. Taken together, the results demonstrate the immense potential of CRISPR/Cas9 based approaches to achieve high frequency targeted edits in regulatory sequences for the generation of novel transcriptional alleles, which may lead to fine tuning of gene expression and functional genomic studies in peanut.

Application of CRISPR/Cas9 System for Efficient Gene Editing in Peanut.

Peanuts are an economically important crop cultivated worldwide. However, several limitations restrained its productivity, including biotic/abiotic stresses. CRISPR/Cas9-based gene-editing technology holds a promising approach to developing new crops with improved agronomic and nutritional traits. Its application has been successful in many important crops. However, the application of this technology in peanut research is limited, probably due to the lack of suitable constructs and protocols. In this study, two different constructs were generated to induce insertion/deletion mutations in the targeted gene for a loss of function study. The first construct harbors the regular gRNA scaffold, while the second construct has the extended scaffold plus terminator. The designed gRNA targeting the coding sequence of the FAD2 genes was cloned into both constructs, and their functionality and efficiency were validated using the hairy root transformation system. Both constructs displayed insertions and deletions as the types of edits. The construct harboring the extended plus gRNA terminator showed a higher editing efficiency than the regular scaffold for monoallelic and biallelic mutations. These two constructs can be used for gene editing in peanuts and could provide tools for improving peanut lines for the benefit of peanut breeders, farmers, and industry.

Genome-Wide Identification of Powdery Mildew Resistance in Common Bean (Phaseolus vulgaris L.).

Genome-wide association studies (GWAS) have been utilized to detect genetic variations related to several agronomic traits and disease resistance in common bean. However, its application in the powdery mildew (PM) disease to identify candidate genes and their location in the common bean genome has not been fully addressed. Single-nucleotide polymorphism (SNP) genotyping with a BeadChip containing 5398 SNPs was used to detect genetic variations related to PM disease resistance in a panel of 211 genotypes grown under two field conditions for two consecutive years. Significant SNPs identified on chromosomes Pv04 and Pv10 were repeatable, ensuring the phenotypic data's reliability and the causal relationship. A cluster of resistance genes was revealed on the Pv04 of the common bean genome, coiled-coil-nucleotide-binding site-leucine-rich repeat (CC-NBS-LRR, CNL), and Toll/interleukin-1 receptor-nucleotide-binding site-leucine-rich repeat type (TIR-NBS-LRR, TNL)-like resistance genes were identified. Furthermore, two resistance genes, Phavu_010G1320001g and Phavu_010G136800g, were also identified on Pv10. Further sequence analysis showed that these genes were homologs to the disease-resistance protein (RLM1A-like) and the putative disease-resistance protein (At4g11170.1) in Arabidopsis. Significant SNPs related to two LRR receptor-like kinases (RLK) were only identified on Pv11 in 2018. Many genes encoding the auxin-responsive protein, TIFY10A protein, growth-regulating factor five-like, ubiquitin-like protein, and cell wall RBR3-like protein related to PM disease resistance were identified nearby significant SNPs. These results suggested that the resistance to PM pathogen involves a network of many genes constitutively co-expressed.

Nicotiana species as surrogate host for studying the pathogenicity of Acidovorax citrulli, the causal agent of bacterial fruit blotch of cucurbits.

Bacterial fruit blotch (BFB) caused by Acidovorax citrulli is one of the most important bacterial diseases of cucurbits worldwide. However, the mechanisms associated with A. citrulli pathogenicity and genetics of host resistance have not been extensively investigated. We idenitfied Nicotiana benthamiana and Nicotiana tabacum as surrogate hosts for studying A. citrulli pathogenicity and non-host resistance triggered by type III secreted (T3S) effectors. Two A. citrulli strains, M6 and AAC00-1, that represent the two major groups amongst A. citrulli populations, induced disease symptoms on N. benthamiana, but triggered a hypersensitive response (HR) on N. tabacum plants. Transient expression of 19 T3S effectors from A. citrulli in N. benthamiana leaves revealed that three effectors, Aave_1548, Aave_2708, and Aave_2166, trigger water-soaking-like cell death in N. benthamiana. Aave_1548 knockout mutants of M6 and AAC00-1 displayed reduced virulence on N. benthamiana and melon (Cucumis melo L.). Transient expression of Aave_1548 and Aave_2166 effectors triggered a non-host HR in N. tabacum, which was dependent on the functionality of the immune signalling component, NtSGT1. Hence, employing Nicotiana species as surrogate hosts for studying A. citrulli pathogenicity may help characterize the function of A. citrulli T3S effectors and facilitate the development of new strategies for BFB management.