Search

Parasitic weeds cause billions of dollars in agricultural losses each year worldwide. Cuscuta campestris (C. campestris), one of the most widespread and destructive parasitic plants in the United States, severely reduces yield in tomato plants. Reducing the spread of parasitic weeds requires understanding the interaction between parasites and hosts. Several studies have identified factors needed for parasitic plant germination and haustorium induction, and genes involved in host defense responses. However, knowledge of the mechanisms underlying the interactions between host and parasitic plants, specifically at the interface between the two organisms, is relatively limited. A detailed investigation of the crosstalk between the host and parasite at the tissue-specific level would enable development of effective parasite control strategies. To focus on the haustorial interface, we used laser-capture microdissection (LCM) with RNA-seq on early, intermediate and mature haustorial stages. In addition, the tomato host tissue that immediately surround the haustoria was collected to obtain tissue- resolution RNA-Seq profiles for C. campestris and tomato at the parasitism interface. After conducting RNA-Seq analysis and constructing gene coexpression networks (GCNs), we identified CcHB7, CcPMEI, and CcERF1 as putative key regulators involved in C. campestris haustorium organogenesis, and three potential regulators, SlPR1, SlCuRe1-like, and SlNLR, in tomatoes that are involved in perceiving signals from the parasite. We used host-induced gene silencing (HIGS) transgenic tomatoes to knock-down the candidate genes in C. campestris and produced CRISPR transgenic tomatoes to knock out candidate genes in tomatoes. The interactions of C. campestris with these transgenic lines were tested and compared with that in wild-type tomatoes. The results of this study reveal the tissue-resolution gene regulatory mechanisms at the parasitic plant-host interface and provide the potential of developing a parasite-resistant system in tomatoes.

Cuscuta species (dodders) are agriculturally destructive, parasitic angiosperms. These parasitic plants use haustoria as physiological bridges to extract nutrients and water from hosts. Cuscuta campestris has a broad host range and wide geographical distribution. While some wild tomato relatives are resistant, cultivated tomatoes are generally susceptible to C. campestris infestations. However, some specific Heinz tomato (Solanum lycopersicum) hybrid cultivars exhibit resistance to dodders in the field, but their defense mechanism was previously unknown. Here, we discovered that the stem cortex in these resistant lines responds with local lignification upon C. campestris attachment, preventing parasite entry into the host. Lignin Induction Factor 1 (LIF1, an AP2-like transcription factor), SlMYB55, and Cuscuta R-gene for Lignin-based Resistance 1, a CC-NBS-LRR (CuRLR1) are identified as factors that confer host resistance by regulating lignification. SlWRKY16 is upregulated upon C. campestris infestation and potentially negatively regulates LIF1 function. Intriguingly, CuRLR1 may play a role in signaling or function as an intracellular receptor for receiving Cuscuta signals or effectors, thereby regulating lignification-based resistance. In summary, these four regulators control the lignin-based resistance response in specific Heinz tomato cultivars, preventing C. campestris from parasitizing resistant tomatoes. This discovery provides a foundation for investigating multilayer resistance against Cuscuta species and has potential for application in other essential crops attacked by parasitic plants.

Parasitic plants reduce crop yield worldwide. Dodder (Cuscuta campestris) is a stem parasite that attaches to its host, using haustoria to extract nutrients and water. We analyzed the transcriptome of six C. campestris tissues and identified a key gene, LATERAL ORGAN BOUNDARIES DOMAIN 25 (CcLBD25), as highly expressed in prehaustoria and haustoria. Gene coexpression networks from different tissue types and laser-capture microdissection RNA-sequencing data indicated that CcLBD25 could be essential for regulating cell wall loosening and organogenesis. We employed host-induced gene silencing by generating transgenic tomato (Solanum lycopersicum) hosts that express hairpin RNAs to target and down-regulate CcLBD25 in the parasite. Our results showed that C. campestris growing on CcLBD25 RNAi transgenic tomatoes transited to the flowering stage earlier and had reduced biomass compared with C. campestris growing on wild-type (WT) hosts, suggesting that parasites growing on transgenic plants were stressed due to insufficient nutrient acquisition. We developed an in vitro haustorium system to assay the number of prehaustoria produced on strands from C. campestris. Cuscuta campestris grown on CcLBD25 RNAi tomatoes produced fewer prehaustoria than those grown on WT tomatoes, indicating that down-regulating CcLBD25 may affect haustorium initiation. Cuscuta campestris haustoria growing on CcLBD25 RNAi tomatoes exhibited reduced pectin digestion and lacked searching hyphae, which interfered with haustorium penetration and formation of vascular connections. The results of this study elucidate the role of CcLBD25 in haustorium development and might contribute to developing parasite-resistant crops.

Parasitic flowering plants are one of the most destructive agricultural pests and have major impact on crop yields throughout the world. Being dependent on finding a host plant for growth, parasitic plants penetrate their host using specialized organs called haustoria. Haustoria establish vascular connections with the host, which enable the parasite to steal nutrients and water. The underlying molecular and developmental basis of parasitism by plants is largely unknown. In order to investigate the process of parasitism, RNAs from different stages (i.e. seed, seedling, vegetative strand, prehaustoria, haustoria, and flower) were used to de novo assemble and annotate the transcriptome of the obligate plant stem parasite dodder (Cuscuta pentagona). The assembled transcriptome was used to dissect transcriptional dynamics during dodder development and parasitism and identified key gene categories involved in the process of plant parasitism. Host plant infection is accompanied by increased expression of parasite genes underlying transport and transporter categories, response to stress and stimuli, as well as genes encoding enzymes involved in cell wall modifications. By contrast, expression of photosynthetic genes is decreased in the dodder infective stages compared with normal stem. In addition, genes relating to biosynthesis, transport, and response of phytohormones, such as auxin, gibberellins, and strigolactone, were differentially expressed in the dodder infective stages compared with stems and seedlings. This analysis sheds light on the transcriptional changes that accompany plant parasitism and will aid in identifying potential gene targets for use in controlling the infestation of crops by parasitic weeds.

Flower scent is a highly dynamic trait, under developmental, spatial, and diurnal regulation. The mechanism governing scent production is only beginning to be unraveled. In petunia (Petunia hybrida), EMISSION OF BENZENOIDS II (EOBII) controls transcription of both the shikimate pathway-regulating MYB factor ODORANT1 (ODO1) and phenylpropanoid scent-related structural genes. A promoter-activation screen identified an R2R3-MYB–like regulatory factor of phenylpropanoid volatile biosynthesis acting downstream of EOBII, designated EOBI. EOBI silencing led to downregulation of ODO1 and numerous structural scent-related genes from both the shikimate and phenylpropanoid pathways. The ability of EOBI to directly activate ODO1, as revealed by electrophoretic mobility shift assay and yeast one-hybrid analysis, place EOBI upstream of ODO1 in regulating substrate availability for volatile biosynthesis. Interestingly, ODO1-silenced transgenic petunia flowers accumulated higher EOBI transcript levels than controls, suggesting a complex feedback loop between these regulatory factors. The accumulation pattern of EOBI transcript relative to EOBII and ODO1, and the effect of up/downregulation of EOBII on transcript levels of EOBI and ODO1, further support these factors' hierarchical relationships. The dependence of scent production on EOBI expression and its direct interaction with both regulatory and structural genes provide evidence for EOBI's wide-ranging involvement in the production of floral volatiles.

Despite a long-standing interest in the genetic basis of morphological diversity, the molecular mechanisms that give rise to developmental variation are incompletely understood. Here, we use comparative transcriptomics coupled with the construction of gene coexpression networks to predict a gene regulatory network (GRN) for leaf development in tomato and two related wild species with strikingly different leaf morphologies. The core network in the leaf developmental GRN contains regulators of leaf morphology that function in global cell proliferation with peripheral gene network modules (GNMs). The BLADE-ON-PETIOLE (BOP) transcription factor in one GNM controls the core network by altering effective concentration of the KNOTTED-like HOMEOBOX gene product. Comparative network analysis and experimental perturbations of BOP levels suggest that variation in BOP expression could explain the diversity in leaf complexity among these species through dynamic rewiring of interactions in the GRN. The peripheral location of the BOP-containing GNM in the leaf developmental GRN and the phenotypic mimics of evolutionary diversity caused by alteration in BOP levels identify a key role for this GNM in canalizing the leaf morphospace by modifying the maturation schedule of leaves to create morphological diversity.

Rose flowers, like flowers and fruits of many other plants, produce and emit the aromatic volatiles 2-phenylacetaldehyde (PAA) and 2-phenylethylalchohol (PEA) which have a distinctive flowery/rose-like scent. Previous studies in rose have shown that, similar to petunia flowers, PAA is formed from L: -phenylalanine via pyridoxal-5'-phosphate-dependent L: -aromatic amino acid decarboxylase. Here we demonstrate the use of a Saccharomyces cerevisiae aro10 mutant to functionally characterize a Rosa hybrida cv. Fragrance Cloud sequence (RhPAAS) homologous to petunia phenylacetaldehyde synthase (PhPAAS). Volatile headspace analysis of the aro10 knockout strain showed that it produces up to eight times less PAA and PEA than the WT. Expression of RhPAAS in aro10 complemented the yeast's mutant phenotype and elevated PAA levels, similar to petunia PhPAAS. PEA production levels were also enhanced in both aro10 and WT strains transformed with RhPAAS, implying an application for metabolic engineering of PEA biosynthesis in yeast. Characterization of spatial and temporal RhPAAS transcript accumulation in rose revealed it to be specific to floral tissues, peaking in mature flowers, i.e., coinciding with floral scent production and essentially identical to other rose scent-related genes. RhPAAS transcript, as well as PAA and PEA production in flowers, displayed a daily rhythmic behavior, reaching peak levels during the late afternoon hours. Examination of oscillation of RhPAAS transcript levels under free-running conditions suggested involvement of the endogenous clock in the regulation of RhPAAS expression in rose flowers.