Novel Sources of Resistance to Fusarium oxysporum f. sp. lycopersici Race 3 Among Solanum pennellii Accessions

Fusarium wilt of tomato (Solanum lycopersicum), caused by fungal pathogen Fusarium oxysporum f. sp. lycopersici (Fol), is one of the most important diseases in tomato production. Three races of the pathogen are described, and race-specific resistance genes have been applied in commercial tomato cultivars for controlling the disease. Race 3 (Fol3) threatens tomato production in many regions around the world, and novel resistance resources could expand the diversity and durability of Fol resistance. The wild tomato species, Solanum pennellii, is reported to harbor broad resistance to Fol and was the source of two known Fol3 resistance genes. In this study, we evaluated 42 S. pennellii accessions for resistance to each fusarium wilt race. F1 plants, developed from crossing each accession with the Fol3 susceptible line ‘Suncoast’, were evaluated for Fol3 resistance, and BC1F1 plants were screened to determine the likelihood that Fol3 resistance was based on a novel locus (loci). Nearly all accessions showed resistance to Fol3, and many accessions were resistant to all races. Evaluation of F1 plants indicated a dominant resistance effect to Fol3 from most accessions. Genetic analysis indicated 24 accessions are expected to contain one or more novel Fol3 resistance loci other than an allele near the I-3 locus. To investigate genetic structure of the S. pennellii accessions used in this study, we genotyped all 42 accessions using genotyping by sequencing. Approximately 20% of the single nucleotide polymorphism (SNP) loci were heterozygous across accessions, likely due to the outcrossing nature of the species. Genetic structure analysis at 49,120 unique SNP loci across accessions identified small but obvious genetic differentiations.

A Cysteine-Rich Protein, SpDIR1L, Implicated in S-RNase-Independent Pollen Rejection in the Tomato (Solanum Section Lycopersicon) Clade

Tomato clade species (Solanum sect. Lycopersicon) display multiple interspecific reproductive barriers (IRBs). Some IRBs conform to the SI x SC rule, which describes unilateral incompatibility (UI) where pollen from SC species is rejected on SI species’ pistils, but reciprocal pollinations are successful. However, SC x SC UI also exists, offering opportunities to identify factors that contribute to S-RNase-independent IRBs. For instance, SC Solanum pennellii LA0716 pistils only permit SC Solanum lycopersicum pollen tubes to penetrate to the top third of the pistil, while S. pennellii pollen penetrates to S. lycopersicum ovaries. We identified candidate S. pennellii LA0716 pistil barrier genes based on expression profiles and published results. CRISPR/Cas9 mutants were created in eight candidate genes, and mutants were assessed for changes in S. lycopersicum pollen tube growth. Mutants in a gene designated Defective in Induced Resistance 1-like (SpDIR1L), which encodes a small cysteine-rich protein, permitted S. lycopersicum pollen tubes to grow to the bottom third of the style. We show that SpDIR1L protein accumulation correlates with IRB strength and that species with weak or no IRBs toward S. lycopersicum pollen share a 150 bp deletion in the upstream region of SpDIR1L. These results suggest that SpDIR1L contributes to an S-RNase-independent IRB.

Characterization of 15-cis-ζ-Carotene Isomerase Z-ISO in Cultivated and Wild Tomato Species Differing in Ripe Fruit Pigmentation

Isomerization of 9,15,9′-tri-cis-ζ-carotene mediated by 15-cis-ζ-carotene isomerase Z-ISO is a critical step in the biosynthesis of carotenoids, which define fruit color. The tomato clade (Solanum section Lycopersicon) comprises the cultivated tomato (Solanum lycopersicum) and 12 related wild species differing in fruit color and, thus, represents a good model for studying carotenogenesis in fleshy fruit. In this study, we identified homologous Z-ISO genes, including 5′-UTRs and promoter regions, in 12 S. lycopersicum cultivars and 5 wild tomato species (red-fruited Solanum pimpinellifolium, yellow-fruited Solanum cheesmaniae, and green-fruited Solanum chilense, Solanum habrochaites, and Solanum pennellii). Z-ISO homologs had a highly conserved structure, suggesting that Z-ISO performs a similar function in tomato species despite the difference in their fruit color. Z-ISO transcription levels positively correlated with the carotenoid content in ripe fruit of the tomatoes. An analysis of the Z-ISO promoter and 5′-UTR sequences revealed over 130 cis-regulatory elements involved in response to light, stresses, and hormones, and in the binding of transcription factors. Green- and red/yellow-fruited Solanum species differed in the number and position of cis-elements, indicating changes in the transcriptional regulation of Z-ISO expression during tomato evolution, which likely contribute to the difference in fruit color.

Physiological and Biochemical Responses of Tomato Plants Grafted onto Solanum pennellii and Solanum peruvianum under Water-deficit Conditions

Grafting using suitable rootstocks mitigates the adverse effects caused by environmental stresses such as water deficit in the tomato crop. Solanum pennellii and Solanum peruvianum, the wild relatives of tomato, are used as rootstocks due to their tolerance to water deficit and soil-borne diseases. This study focused on evaluating physiological and biochemical responses of tomato plants grafted onto S. pennellii and S. peruvianum rootstocks during water deficit. The commercial tomato cultivar ‘HM 1823′ (HM) either self-grafted (HM/HM) or grafted onto S. pennellii (HM/PN), S. peruvianum (HM/PR), and ‘Multifort’ (HM/MU) rootstocks were subjected to water-deficit stress by withholding irrigation for eight days. The performance of the grafted plants under water deficit was evaluated using physiological and biochemical parameters in vegetative tissues of the grafted plants. Plants grafted using S. pennellii (PN) and S. peruvianum (PR) rootstocks showed higher values of water potential (Ѱw), relative water content (RWC), net photosynthetic rate (A), and leaf water use efficiencies (WUE) compared to HM, HM/HM, and HM/MU. Plants grafted onto tomato wild relatives showed the lowest malondialdehyde (MDA) and proline content. This study demonstrated that the rootstocks of wild tomato relatives reduced the effect of water deficit to a greater extent through better physiological, metabolic, and biochemical adjustments than self-grafting plants.

In silico analysis and molecular investigation of the nuclear shuttle protein-interacting kinase 1 (NIK1) in six Solanaceous species

The first layer of innate immunity in plants is initiated through the perception of microbe-associated molecular patterns (MAMPs) or damage-associated molecular patterns (DAMPs) by pattern recognition receptors (PRRs). MAMP/DAMP perception initiates downstream defense responses, a process which ultimately leads to pattern triggered immunity (PTI). In Arabidopsis, the nuclear shuttle protein-interacting kinase 1 (NIK1), among other PRRs, is one of the most important central components of PTI signaling and kinase signaling cascade, since it is involved in the plant antiviral response against geminiviruses. Despite the characterization of the structure and function of the NIK1 receptors made by some groups, studies related to NIK1 importance in the current gene-editing era are missing. By simple in silico analysis, in this study we investigated the NIK1 homologues from six Solanaceous plant species including: tomato (Solanum lycopersicum), potato (Solanum tuberosum), Solanum pennellii, eggplant (Solanum melongena), pepper (Capsicum annum), and Nicotiana benthamiana. The phylogenetic analyses of different NIK1 proteins from Arabidopsis and six Solanaceous plants revealed nine different clades. As expected, we found that these NIK1 orthologs have similar genomic structures suggesting a similar function. We could identify that SotubNIK1, SolyNIK1, SopenNIK1, CANIK1, NibenNIK1, SmeNIK1 have the highest sequence homology with AtNIK1. Additionally, the conserved protein kinase domain (PKD) that is present in NIK1 from Arabidopsis thaliana was bioinformatically analyzed and found in other species. As this highly conserved NIK1 region is present in several crops of economic importance, its potential is highlighted as a possible target site for gene editing, to develop crops tolerant to geminiviral infections.

Basic Analysis of Calcium-dependent Protein Kinase Gene and Its Closely Related Gene Families in Solanum Pennellii Genome

Calcium-dependent protein kinases (CDPK) are the main Ca2 + sensor involved in the regulation of plant growth and development and various stress responses. In this study, we identified 32 CDPK (SpCDPK) genes and 7 CDPK-related protein kinase (SpCRK) genes in the whole genome of Solanum Pennellii, which were unevenly distributed on 12 chromosomes. The SpCDPK and SpCRK proteins own ATP-binding region and Ser/Thr protein kinase region. However, the SpCDPK proteins had EF-hand calcium-binding region, but the SpCRK proteins lacked it. Phylogenetic analysis showed that the SpCDPK and SpCRK gene families in Solanum Pennellii could be divided into four subgroups, and the evolutionary relationship between Solanum Pennellii and Arabidopsis thaliana was closer. Further analysis revealed that the exon-intron structure and conserved motif of each subgroup were basically the same, but there were differences in cis-acting elements. In this study, we conducted a preliminary analysis of SpCDPK and SpCRK gene families in Solanum Pennellii to provide basic data for further exploration of its molecular mechanism.

The ORGAN SIZE (ORG) locus contributes to isometric gigantism in domesticated tomato

Gigantism is a key component of the domestication syndrome, a suite of traits that differentiates crops from their wild relatives. Allometric gigantism is strongly marked in horticultural crops, causing disproportionate increases in the size of edible parts such as stems, leaves or fruits. Tomato (Solanum lycopersicum) has attracted attention as a model for fruit gigantism, and many genes have been described controlling this trait. However, the genetic basis of a corresponding increase in size of vegetative organs contributing to isometric gigantism, has remained relatively unexplored. Here, we identified a 0.4 Mbp region on chromosome 7 in introgression lines (ILs) from the wild species Solanum pennellii in two different tomato genetic backgrounds (cv. M82 and cv. Micro-Tom) that controls vegetative and reproductive organ size in tomato. The locus, named ORGAN SIZE (ORG), was fine-mapped using genotype-by-sequencing. A survey of literature revealed that ORG overlaps with previously mapped QTLs controlling tomato fruit weight during domestication. Alleles from the wild species led to reduced cell number in different organs, which was partially compensated by greater cell expansion in leaves but not in fruits. The result was a proportional reduction in leaf, flower and fruit size in the ILs harbouring the wild alleles. These findings suggest that selection for large fruit during domestication also tends to select for increases in leaf size by influencing cell division. Since leaf size is relevant for both source-sink balance and crop adaptation to different environments, the discovery of ORG could allow fine-tuning of these parameters.