Comparative secretome analysis of Striga and Cuscuta species identifies candidate virulence factors for two evolutionarily independent parasitic plant lineages.

BACKGROUND: Many parasitic plants of the genera Striga and Cuscuta inflict huge agricultural damage worldwide. To form and maintain a connection with a host plant, parasitic plants deploy virulence factors (VFs) that interact with host biology. They possess a secretome that represents the complement of proteins secreted from cells and like other plant parasites such as fungi, bacteria or nematodes, some secreted proteins represent VFs crucial to successful host colonisation. Understanding the genome-wide complement of putative secreted proteins from parasitic plants, and their expression during host invasion, will advance understanding of virulence mechanisms used by parasitic plants to suppress/evade host immune responses and to establish and maintain a parasite-host interaction. RESULTS: We conducted a comparative analysis of the secretomes of root (Striga spp.) and shoot (Cuscuta spp.) parasitic plants, to enable prediction of candidate VFs. Using orthogroup clustering and protein domain analyses we identified gene families/functional annotations common to both Striga and Cuscuta species that were not present in their closest non-parasitic relatives (e.g. strictosidine synthase like enzymes), or specific to either the Striga or Cuscuta secretomes. For example, Striga secretomes were strongly associated with 'PAR1' protein domains. These were rare in the Cuscuta secretomes but an abundance of 'GMC oxidoreductase' domains were found, that were not present in the Striga secretomes. We then conducted transcriptional profiling of genes encoding putatively secreted proteins for the most agriculturally damaging root parasitic weed of cereals, S. hermonthica. A significant portion of the Striga-specific secretome set was differentially expressed during parasitism, which we probed further to identify genes following a 'wave-like' expression pattern peaking in the early penetration stage of infection. We identified 39 genes encoding putative VFs with functions such as cell wall modification, immune suppression, protease, kinase, or peroxidase activities, that are excellent candidates for future functional studies. CONCLUSIONS: Our study represents a comprehensive secretome analysis among parasitic plants and revealed both similarities and differences in candidate VFs between Striga and Cuscuta species. This knowledge is crucial for the development of new management strategies and delaying the evolution of virulence in parasitic weeds.

Abscisic acid inhibits germination of Striga seeds and is released by them likely as a rhizospheric signal supporting host infestation.

Seeds of the root parasitic plant Striga hermonthica undergo a conditioning process under humid and warm environments before germinating in response to host-released stimulants, particularly strigolactones (SLs). The plant hormone abscisic acid (ABA) regulates different growth and developmental processes, and stress response; however, its role during Striga seed germination and early interactions with host plants is under-investigated. Here, we show that ABA inhibited Striga seed germination and that hindering its biosynthesis induced conditioning and germination in unconditioned seeds, which was significantly enhanced by treatment with the SL analog rac-GR24. However, the inhibitory effect of ABA remarkably decreased during conditioning, confirming the loss of sensitivity towards ABA in later developmental stages. ABA measurement showed a substantial reduction of its content during the early conditioning stage and a significant increase upon rac-GR24-triggered germination. We observed this increase also in released seed exudates, which was further confirmed by using the Arabidopsis ABA-reporter GUS marker line. Seed exudates of germinated seeds, containing elevated levels of ABA, impaired the germination of surrounding Striga seeds in vitro and promoted root growth of a rice host towards germinated Striga seeds. Application of ABA as a positive control caused similar effects, indicating its function in Striga/Striga and Striga/host communications. In summary, we show that ABA is an essential player during seed dormancy and germination processes in Striga and acts as a rhizospheric signal likely to support host infestation.

A microbial bioherbicide for Striga hermonthica control: production, development, and effectiveness of a seed coating agent.

Witchweed (Striga hermonthica), also called striga, is a parasitic weed that causes high yield losses in maize on more than 200 000 ha in Kenya alone. A new commercial, biological herbicide developed in Kenya is able to control striga effectively. The product was approved for use by the Pest Control Products Board in Kenya in September, 2021. It is self-produced in villages using a secondary inoculum provided by a commercial company. The formulated product has some disadvantages, which are a complicated production process, a very short shelf life and high application rate. Additionally, the product has to be applied manually and therefore can only be used in manual production, leaving out the opportunity for farmers using mechanization. For this reason, efforts have been made to formulate the active ingredient Fusarium oxysporum f. sp. strigae strain DSM 33471, as a powder and to use it as a seed coating agent. This article deals with the production of the Fusarium spore powder, its properties, its application to the seed, and its herbicidal effect demonstrated in the first two field trials. The F. oxysporum strain was originally isolated from a wilting striga plant in Kenya. The strain was virulence enhanced to over produce the amino acids leucine, methionine and tyrosine. These amino acids are responsible for a second mode of action apart from the wilting causing effect of the fungus on striga. Whereas leucine and tyrosine have a herbicidal effect, ethylene from methionine triggers the germination of striga seeds in the soil. Additionally, the strain has been improved to be resistant to the fungicide captan, which is commonly used to treat maize seed in Kenya. Seed coating tests conducted on 25 striga-infested small holder farms spread out in six counties of western Kenya reported yield increases of up to 88%. A second trial carried out by the Kenyan Agricultural and Livestock Research Organization showed a 93% reduction of emerged striga plants. © 2023 Society of Chemical Industry.

Why are there no widely successful microbial bioherbicides for weed management in crops?

Microbial biopesticides to control plant pathogens and insects in crops have had significant success. However, there have been relatively few successes for microbial bioherbicides in crops, despite considerable numbers of publications and commercial product introductions in this area. Marketed microbial bioherbicide products for use in agriculture have been largely unsuccessful. This article covers the potential advantages of successful microbial bioherbicides, as well as the biological and technical issues that have limited their success. Technologies to overcome the problems that have limited the success of these products are discussed. The many advantages of using killed microbial products (e.g. cell-free filtrates) over living microbial products as bioherbicides are detailed. A commercialized mycoherbicide that has been selected for in the laboratory for control of the parasitic weed Striga hermonthica is being used with some success in Africa, indicating that non-transgenic modification of the genetics of bioherbicide microbes for improved efficacy is acceptable to some regulatory authorities. Genetic modifications to improve efficacy and host range, as well as improved application technology to greatly reduce the amount of product needed are two technologies that are likely to expand the use of microbial bioherbicides in the future. © 2023 Society of Chemical Industry.

The Toothpick Project: commercialization of a virulence-selected fungal bioherbicide for Striga hermonthica (witchweed) biocontrol in Kenya.

The high-level view of global food systems identifies three all-encompassing barriers to the adoption of food systems solutions: knowledge, policy, and finance. These barriers, and the siloed characteristics of each of these, have hindered the development and adoption of microbial herbicides. How knowledge, policy, and finance are related to the Toothpick Project's path of commercializing a new bioherbicide, early in the scope of the industry, is discussed here. The Toothpick Project's innovation, developed over four decades and commercialized in 2021, uses strains of Fusarium oxysporum f.sp. strigae selected for overproduction and excretion of specific amino acids, killing the parasitic weed Striga hermonthica (Striga or witchweed), Africa's worst pest threat to food security. Historically, bioherbicides have not been a sufficient alternative to the dominant use of synthetic chemical herbicides. To be used safely as bioherbicides, plant pathogens need to be host specific, non-toxic, and yet sufficiently virulent to control a specific weed. For commercialization, bioherbicides must be affordable and require a sufficient shelf life for distribution. Given the current triple storm encountered by the chemical herbicide industry (herbicide-resistant weeds, lawsuits, and consumer pushback), there exists an opportunity to use certain plant pathogens as bioherbicides by enhancing their virulence. By discussing barriers in the scope of knowledge, policy, and finance in the development of the Toothpick Project's new microbial bioherbicide, we hope to help others to anticipate the challenges and provide change-leaders, particularly in policy and finance, a ground level perspective of bioherbicide development. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Enhancing Genetic Gains in Grain Yield and Efficiency of Testing Sites of Early-Maturing Maize Hybrids under Contrasting Environments.

The major challenges of maize production and productivity in Sub-Saharan Africa (SSA) include Striga hermonthica infestation, recurrent drought, and low soil nitrogen (low N). This study assessed the following: (i) accelerated genetic advancements in grain yield and other measured traits of early-maturing maize hybrids, (ii) ideal test environments for selecting early-maturing multiple-stress tolerant hybrids, and (iii) high-yielding and stable hybrids across multiple-stress and non-stress environments. Fifty-four hybrids developed during three periods of genetic enhancement (2008-2010, 2011-2013, and 2014-2016) were evaluated in Nigeria, The Republic of Benin, and Ghana under multiple stressors (Striga infestation, managed drought, and Low N) and non-stress environments from 2017 to 2019. Under multiple-stress and non-stress environments, annual genetic gains from selection in grain yield of 84.72 kg ha-1 (4.05%) and 61 kg ha-1 (1.56%), respectively, were recorded. Three mega-environments were identified across 14 stress environments. Abuja was identified as an ideal test environment for selecting superior hybrids. The hybrid TZdEI 352 × TZEI 355 developed during period 3 was the most outstanding under multiple-stress and non-stress environments. On-farm testing of this hybrid is required to verify its superior performance for commercialization in SSA. Considerable progress has been made in the genetic improvement of early-maturing maize hybrids for tolerance of multiple stressors and high yield. The identified core testing sites of this study could be used to enhance the testing and selection of promising hybrids.

Hydroxamic acids: New players in the multifactorial mechanisms of maize resistance to Striga hermonthica.

Striga hermonthica is the most widespread and destructive plant parasite infesting maize and other major crops in sub-Saharan Africa where it causes severe yield losses and threatens food security. Several tolerant maize lines supporting reduced S. hermonthica emergence have been deployed. However, the molecular bases of such resistance are yet poorly understood. Based on a time course comparative gene expression analysis between susceptible and resistant maize lines we have confirmed resistance mechanisms known to be activated upon plant parasite infestation and identified potential novel players worth further investigation e.g. iron homeostasis and mitochondrial respiration-related genes. Most intriguingly, we show a previously unknown strategy of maize post-attachment resistance based on DIMBOA accumulation in S. hermonthica-infested maize roots. S. hermonthica infestation triggers positive regulation of gene expression in the hydroxamic acid (HA) pathway culminating with an accumulation of benzoxazinoids (BX), known for their antifeedant, insecticidal, antimicrobial, and allelopathic activities. We demonstrate that HA root content is positively correlated with S. hermonthica resistance in the resistant parent and its progenies and in unrelated maize lines. Downregulation of HA genes causes increased susceptibility to S. hermonthica infestation in loss-of-function maize mutants. While the mechanism of BX action in parasitic plant resistance is yet to be uncovered, the potential of this discovery for developing effective control and breeding strategies is enormous.

Genome-wide association analyses of agronomic traits and Striga hermonthica resistance in pearl millet.

Pearl millet (Pennisetum glaucum [L.] R. Br.) is a nutrient-dense, relatively drought-tolerant cereal crop cultivated in dry regions worldwide. The crop is under-researched, and its grain yield is low (< 0.8 tons ha-1) and stagnant in the major production regions, including Burkina Faso. The low productivity of pearl millet is mainly attributable to a lack of improved varieties, Striga hermonthica [Sh] infestation, downy mildew infection, and recurrent heat and drought stress. Developing high-yielding and Striga-resistant pearl millet varieties that satisfy the farmers' and market needs requires the identification of yield-promoting genes linked to economic traits to facilitate marker-assisted selection and gene pyramiding. The objective of this study was to undertake genome-wide association analyses of agronomic traits and Sh resistance among 150 pearl millet genotypes to identify genetic markers for marker-assisted breeding and trait introgression. The pearl millet genotypes were phenotyped in Sh hotspot fields and screen house conditions. Twenty-nine million single nucleotide polymorphisms (SNPs) initially generated from 345 pearl millet genotypes were filtered, and 256 K SNPs were selected and used in the present study. Phenotypic data were collected on days to flowering, plant height, number of tillers, panicle length, panicle weight, thousand-grain weight, grain weight, number of emerged Striga and area under the Striga number progress curve (ASNPC). Agronomic and Sh parameters were subjected to combined analysis of variance, while genome-wide association analysis was performed on phenotypic and SNPs data. Significant differences (P < 0.001) were detected among the assessed pearl millet genotypes for Sh parameters and agronomic traits. Further, there were significant genotype by Sh interaction for the number of Sh and ASNPC. Twenty-eight SNPs were significantly associated with a low number of emerged Sh located on chromosomes 1, 2, 3, 4, 6, and 7. Four SNPs were associated with days-to-50%-flowering on chromosomes 3, 5, 6, and 7, while five were associated with panicle length on chromosomes 2, 3, and 4. Seven SNPs were linked to thousand-grain weight on chromosomes 2, 3, and 6. The putative SNP markers associated with a low number of emerged Sh and agronomic traits in the assessed genotypes are valuable genomic resources for accelerated breeding and variety deployment of pearl millet with Sh resistance and farmer- and market-preferred agronomic traits.

A roadmap of haustorium morphogenesis in parasitic plants.

Parasitic plants invade their host through their invasive organ, the haustorium. This organ connects to the vasculature of the host roots and hijacks water and nutrients. Although parasitism has evolved independently in plants, haustoria formation follows a similar mechanism throughout different plant species, highlighting the developmental plasticity of plant tissues. Here, we compare three types of haustoria formed by the root and shoot in the plant parasites Striga and Cuscuta. We discuss mechanisms underlying the interactions with their hosts and how different approaches have contributed to major understanding of haustoria formation and host invasion. We also illustrate the role of auxin and cytokinin in controlling this process.

Role of Strigolactones in the Host Specificity of Broomrapes and Witchweeds.

Root parasitic plants of the Orobanchaceae, broomrapes and witchweeds, pose a severe problem to agriculture in Europe, Asia and especially Africa. These parasites are totally dependent on their host for survival, and therefore, their germination is tightly regulated by host presence. Indeed, their seeds remain dormant in the soil until a host root is detected through compounds called germination stimulants. Strigolactones (SLs) are the most important class of germination stimulants. They play an important role in planta as a phytohormone and, upon exudation from the root, function in the recruitment of symbiotic arbuscular mycorrhizal fungi. Plants exude mixtures of various different SLs, possibly to evade detection by these parasites and still recruit symbionts. Vice versa, parasitic plants must only respond to the SL composition that is exuded by their host, or else risk germination in the presence of non-hosts. Therefore, parasitic plants have evolved an entire clade of SL receptors, called HTL/KAI2s, to perceive the SL cues. It has been demonstrated that these receptors each have a distinct sensitivity and specificity to the different known SLs, which possibly allows them to recognize the SL-blend characteristic of their host. In this review, we will discuss the molecular basis of SL sensitivity and specificity in these parasitic plants through HTL/KAI2s and review the evidence that these receptors contribute to host specificity of parasitic plants.

Resolving intergenotypic Striga resistance in sorghum.

Genetic underpinnings of host-pathogen interactions in the parasitic plant Striga hermonthica, a root parasitic plant that ravages cereals in sub-Saharan Africa, are unclear. We performed a comparative transcriptome study on five genotypes of sorghum exhibiting diverse resistance responses to S. hermonthica using weighted gene co-expression network analysis (WGCNA). We found that S. hermonthica elicits both basal and effector-triggered immunity-like a bona fide pathogen. The resistance response was genotype specific. Some resistance responses followed the salicylic acid-dependent signaling pathway for systemic acquired resistance characterized by cell wall reinforcements, lignification, and callose deposition, while in others the WRKY-dependent signaling pathway was activated, leading to a hypersensitive response. In some genotypes, both modes of resistance were activated, while in others either mode dominated the resistance response. Cell wall-based resistance was common to all sorghum genotypes but strongest in IS2814, while a hypersensitive response was specific to N13, IS9830, and IS41724. WGCNA further allowed for pinpointing of S. hermonthica resistance causative genes in sorghum, including glucan synthase-like 10 gene, a pathogenesis-related thaumatin-like family gene, and a phosphoinositide phosphatase gene. Such candidate genes will form a good basis for subsequent functional validation and possibly future resistance breeding.

Gibberellins Promote Seed Conditioning by Up-Regulating Strigolactone Receptors in the Parasitic Plant Striga hermonthica.

Dormant seeds of the root parasitic plant Striga hermonthica sense strigolactones from host plants as environmental cues for germination. This process is mediated by a diversified member of the strigolactone receptors encoded by HYPOSENSITIVE TO LIGHT/KARRIKIN INSENSITIVE2 genes. It is known that warm and moist treatment during seed conditioning gradually makes dormant Striga seeds competent to respond to strigolactones, although the mechanism behind it is poorly understood. In this report, we show that plant hormone gibberellins increase strigolactone competence by up-regulating mRNA expression of the major strigolactone receptors during the conditioning period. This idea was supported by a poor germination phenotype in which gibberellin biosynthesis was depleted by paclobutrazol during conditioning. Moreover, live imaging with a fluorogenic strigolactone mimic, yoshimulactone green W, revealed that paclobutrazol treatment during conditioning caused aberrant dynamics of strigolactone perception after germination. These observations revealed an indirect role of gibberellins in seed germination in Striga, which contrasts with their roles as dominant germination-stimulating hormones in non-parasitic plants. We propose a model of how the role of gibberellins became indirect during the evolution of parasitism in plants. Our work also highlights the potential role for gibberellins in field applications, for instance, in elevating the sensitivity of seeds toward strigolactones in the current suicidal germination approach to alleviate the agricultural threats caused by this parasite in Africa.

Structural analysis of a hormone-bound Striga strigolactone receptor.

Strigolactones (SLs) regulate many aspects of plant development, but ambiguities remain about how this hormone is perceived because SL-complexed receptor structures do not exist. We find that when SL binds the Striga receptor, ShHTL5, a series of conformational changes relative to the unbound state occur, but these events are not sufficient for signalling. Ligand-complexed receptors, however, form internal tunnels that posit an explanation for how SL exits its receptor after hydrolysis.

Conferring resistance to parasitic witchweed by shifting strigolactone biosynthesis.

Strigolactones from the exudates of maize root induce germination of the parasitic witchweed Striga. Recently, Li et al. characterized the biosynthesis pathway of two strigolactones, zealactol and zealactonoic acid, which induce less Striga germination than the major maize strigolactone, zealactone. This study provides a promising strategy for plant protection against parasitic witchweed.

Organosulfur Compound Identified from Striga angustifolia (D. Don) C.J. Saldanha Inhibits Lung Cancer Growth and Induces Apoptosis via p53/mTOR Signaling Pathway.

The Striga angustifolia (D. Don) C.J. Saldanha was used as an Ayurvedic and homeopathic medicine for cancer by the tribal peoples of the Maruthamalai Hills, Coimbatore, India. Hence, the traditional use that has been proven to be effective lacks convincing scientific references. This present study was conducted to investigate the presence of potentially bioactive compounds from S. angustifolia and provides a scientific basis for the ethnobotanical utility. The organosulfur compound 5,5'-dithiobis(1-phenyl-1H-tetrazole) (COMP1) was isolated from S. angustifolia extracts, and the structures of COMP1 were elucidated and characterized by using 13C and 1H nuclear magnetic resonance (NMR) and single crystal X-ray powder diffraction (XRD). Our findings showed that COMP1 significantly reduced cell proliferation of breast and lung cancer cells, but not that of non-malignant epithelial cells. Further analysis revealed that COMP1 promoted cell cycle arrest and apoptosis of lung cancer cells. Mechanistically, COMP1 facilitates p53 activity and inhibits mammalian target of rapamycin (mTOR) signaling, thereby inducing cell cycle arrest and apoptosis of lung cancer cells by inhibiting cell growth. Our findings suggest that COMP1 may serve as a potential drug for lung cancer through the regulation of p53/mTOR pathways.

Combining ability of extra-early maturing pro-vitamin A maize (Zea mays L.) inbred lines and performance of derived hybrids under Striga hermonthica infestation and low soil nitrogen.

Low soil nitrogen (low-N), Striga hermonthica infestation and vitamin A deficiency in normal endosperm maize are major challenges confronting maize production and nutrition of the people of sub-Saharan Africa (SSA). Development of pro-vitamin A (PVA) maize hybrids with combined resistance/tolerance to the two stress factors is crucial in mitigating the food insecurity and nutrition challenges resulting from low-N deficiency and Striga infestation. One hundred and fifty hybrids plus six hybrid checks were evaluated under low-N, Striga-infested and optimal conditions in Nigeria for two years. The study examined the combining ability of the PVA inbreds in contrasting environments, classified them into heterotic groups, examined the inter-relationships of grain yield and other agronomic traits of the inbreds in hybrid combinations and assessed the performance and stability of the PVA hybrids across environments. Additive gene action conditioned the inheritance of grain yield under Striga infestation and optimal conditions while non-additive gene action played the major role in the inheritance of grain yield under low-N. Hybrids TZEEIOR 217 × TZEEIOR 197 and TZEEIOR 245 × TZEEIOR 195 were the top-yielding under Striga and low-N conditions, respectively. Inbred lines were classified into three heterotic groups. Inbreds TZEEIOR 195 and TZEEIOR 221 were identified as testers while TZEEIOR 197 × TZEEIOR 82, TZEEIOR 132 × TZEEIOR 195 and TZEEIOR 205 × TZEEIOR 221 were identified as single-cross testers. Ear aspect had direct contribution to grain yield, justifying its inclusion in the multiple trait base index used for selection of Striga resistant and low-N tolerant maize genotypes in SSA. Hybrids TZEEIOR 195 × TZEEIOR 149, TZEEIOR 195 × TZEEIOR 150, TZEEIOR 245 × TZEEIOR 195, TZEEIOR 30 × TZEEIOR 150 and TZEEIOR 245 × TZEEIOR 30 were high-yielding and stable across test environments. These hybrids should be tested extensively in on-farm trials and commercialized to contribute to food security in SSA.

Maize resistance to witchweed through changes in strigolactone biosynthesis.

Maize (Zea mays) is a major staple crop in Africa, where its yield and the livelihood of millions are compromised by the parasitic witchweed Striga. Germination of Striga is induced by strigolactones exuded from maize roots into the rhizosphere. In a maize germplasm collection, we identified two strigolactones, zealactol and zealactonoic acid, which stimulate less Striga germination than the major maize strigolactone, zealactone. We then showed that a single cytochrome P450, ZmCYP706C37, catalyzes a series of oxidative steps in the maize-strigolactone biosynthetic pathway. Reduction in activity of this enzyme and two others involved in the pathway, ZmMAX1b and ZmCLAMT1, can change strigolactone composition and reduce Striga germination and infection. These results offer prospects for breeding Striga-resistant maize.

Genome-wide association studies of Striga resistance in extra-early maturing quality protein maize inbred lines.

Identification of genes associated with Striga resistance is invaluable for accelerating genetic gains in breeding for Striga resistance in maize. We conducted a genome-wide association study to identify genomic regions associated with grain yield and other agronomic traits under artificial Striga field infestation. One hundred and forty-one extra-early quality protein maize inbred lines were phenotyped for key agronomic traits. The inbred lines were also genotyped using 49,185 DArTseq markers from which 8,143 were retained for population structure analysis and genome wide-association study. Cluster analysis and population structure revealed the presence of 3 well-defined genetic groups. Using the mixed linear model, 22 SNP markers were identified to be significantly associated with grain yield, Striga damage at 10 weeks after planting, number of emerged Striga plants at 8 and 10 weeks after planting and ear aspect. The identified SNP markers would be useful for breeders for marker-assisted selection to accelerate the genetic enhancement of maize for Striga resistance in sub-Saharan Africa after validation.