Phytocytokine StPep1-secreting bacteria suppress potato powdery scab disease.

Powdery scab is an important potato disease caused by the soilborne pathogen Spongospora subterranea f. sp. subterranea. Currently, reliable chemical control and resistant cultivars for powdery scab are unavailable. As an alternative control strategy, we propose a novel approach involving the effective delivery of a phytocytokine to plant roots by the rhizobacterium Bacillus subtilis. The modified strain is designed to secrete the plant elicitor peptide StPep1. In our experiments employing a hairy root system, we observed a significant reduction in powdery scab pathogen infection when directly applying the StPep1 peptide. Furthermore, our pot assay, which involved pretreating potato roots with StPep1-secreting B. subtilis, demonstrated a substantial decrease in disease symptoms, including reduced root galling and fewer tuber skin scabs. These findings underscore the potential of engineered bacteria as a promising strategy for safeguarding plants against powdery scab.

The effects of Bacillus subtilis expressing a plant elicitor peptide on nematode infection on soybean.

There is a pressing need to develop alternative management strategies for the soybean-cyst nematode (Heterodera glycines, SCN), the most costly pathogen to soybeans. Plant elicitor peptides (PEPs), which are produced by plants in response to stress and stimulate broad-spectrum disease resistance, were previously shown to reduce SCN infection on soybeans when applied as a seed treatment. Here, we introduce an alternative method to deliver PEPs to soybean using a common plant growth-promoting rhizobacterium, Bacillus subtilis, as a bacterial expression system. Similar to the empty vector control, B. subtilis engineered to express a PEP from soybean (GmPEP3) was able to colonize soybean roots and persisted on roots more than a month after treatment. Compared to water or the empty vector control, plants that received a seed treatment with B. subtilis expressing GmPEP3 (B.+GmPEP3) were significantly taller early in vegetative growth (V1 stage) and had lower chlorophyll content in the reproductive stage (R3/R4); these results suggested that GmPEP3 may hasten growth and subsequent senescence. When plants were inoculated with SCN at the V1 stage, those pre-treated with B.+GmPEP3 supported significantly fewer nematode eggs at the reproductive stage (R3/R4) than plants treated with water or the empty vector. The effects of B.+GmPEP3 on nematode infection and plant growth appeared to be due primarily to the peptide itself because no significant differences were observed between plants treated with water or with B. subtilis expressing the empty vector. These results indicate the ability of B. subtilis to deliver defense activators for nematode management on soybean.

Unveiling the Diversity: Plant Parasitic Nematode Effectors and Their Plant Interaction Partners.

Root-knot and cyst nematodes are two groups of plant parasitic nematodes that cause the majority of crop losses in agriculture. As a result, these nematodes are the focus of most nematode effector research. Root-knot and cyst nematode effectors are defined as secreted molecules, typically proteins, with crucial roles in nematode parasitism. There are likely hundreds of secreted effector molecules exuded through the nematode stylet into the plant. The current research has shown that nematode effectors can target a variety of host proteins and have impacts that include the suppression of plant immune responses and the manipulation of host hormone signaling. The discovery of effectors that localize to the nucleus indicates that the nematodes can directly modulate host gene expression for cellular reprogramming during feeding site formation. In addition, plant peptide mimicry by some nematode effectors highlights the sophisticated strategies the nematodes employ to manipulate host processes. Here we describe research on the interactions between nematode effectors and host proteins that will provide insights into the molecular mechanisms underpinning plant-nematode interactions. By identifying the host proteins and pathways that are targeted by root-knot and cyst nematode effectors, scientists can gain a better understanding of how nematodes establish feeding sites and subvert plant immune responses. Such information will be invaluable for future engineering of nematode-resistant crops, ultimately fostering advancements in agricultural practices and crop protection. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2024.

A molecular beacon real-time polymerase chain reaction assay for the identification of M. chitwoodi, M. fallax, and M. minor.

Root-knot nematodes (Meloidogyne spp.) are major pests of many important crops around the world. In the Northwestern region of the United States of America (USA), Meloidogyne chitwoodi causes economic losses in potatoes because the nematodes can infect the tubers, which leads to potato galling and reductions in marketable yield. Meloidogyne chitwoodi is a quarantine pathogen in certain potato export markets, and there is little industry tolerance for the presence of this nematode. Recently, two Meloidogyne species that are not known to be present in agricultural fields in the USA were detected on golf turfgrasses in California and Washington. These species, M. fallax and M. minor, are morphologically similar to M. chitwoodi and can infect potatoes and cause tuber damage. Their detection in the USA means that they could potentially infest potato fields and become a problem in potato production. Additionally, M. fallax is a regulated plant pest in the USA, which makes the correct identification of potato-infecting root-knot nematodes important. Previously, there was no single-tube assay that could determine whether M. chitwoodi, M. fallax, and/or M. minor were present in a sample. Thus, a molecular beacon real-time PCR assay which can reliably detect M. chitwoodi, M. fallax, or M. minor from crude nematode extracts was designed and characterized.

Calcium/calmodulin-mediated microbial symbiotic interactions in plants.

Cytoplasmic calcium (Ca2+) transients and nuclear Ca2+ oscillations act as hubs during root nodulation and arbuscular mycorrhizal symbioses. Plants perceive bacterial Nod factors or fungal signals to induce the Ca2+ oscillation in the nucleus of root hair cells, and subsequently activate calmodulin (CaM) and Ca2+/CaM-dependent protein kinase (CCaMK). Ca2+ and CaM-bound CCaMK phosphorylate transcription factors then initiate down-stream signaling events. In addition, distinct Ca2+ signatures are activated at different symbiotic stages: microbial colonization and infection; nodule formation; and mycorrhizal development. Ca2+ acts as a key signal that regulates a complex interplay of downstream responses in many biological processes. This short review focuses on advances in Ca2+ signaling-regulated symbiotic events. It is meant to be an introduction to readers in and outside the field of bacterial and fungal symbioses. We summarize the molecular mechanisms underlying Ca2+/CaM-mediated signaling in fine-tuning both local and systemic symbiotic events.

Rooting Out the Mechanisms of Root-Knot Nematode-Plant Interactions.

Root-knot nematodes (RKNs; Meloidogyne spp.) engage in complex parasitic interactions with many different host plants around the world, initiating elaborate feeding sites and disrupting host root architecture. Although RKNs have been the focus of research for many decades, new molecular tools have provided useful insights into the biological mechanisms these pests use to infect and manipulate their hosts. From identifying host defense mechanisms underlying resistance to RKNs to characterizing nematode effectors that alter host cellular functions, the past decade of research has significantly expanded our understanding of RKN-plant interactions, and the increasing number of quality parasite and host genomes promises to enhance future research efforts into RKNs. In this review, we have highlighted recent discoveries, summarized the current understanding within the field, and provided links to new and useful resources for researchers. Our goal is to offer insights and tools to support the study of molecular RKN-plant interactions.

Transcriptome Analyses of Pre-parasitic and Parasitic Meloidogyne Chitwoodi Race 1 to Identify Putative Effector Genes.

Meloidogyne chitwoodi is a root-knot nematode that is a major pest of potato in the northwestern United States. Due to the lack of resistance against root-knot nematodes in potato, research has been undertaken to understand the M. chitwoodi-potato interaction at the molecular level. To identify the nematode genes that are playing roles in parasitism, we have performed transcriptome analyses on pre-parasitic and parasitic M. chitwoodi juveniles in susceptible potato. We compared gene expression profiles and identified genes that were significantly up- or down-regulated during nematode parasitism. Because parasitism proteins are typically secreted by the nematode to facilitate infection of host roots, we focused on the genes that encoded proteins that were predicted to be secreted. We found that approximately 34% (43/127) of the genes in the predicted secretome encoded proteins with no significant homology in the public genome databases, and 12% (15/127) encoded either a known effector, putative effectors or putative esophageal gland cell proteins. The transcriptome analyses of M. chitwoodi at the pre-parasitic and parasitic life stages shed light on the genes involved in nematode parasitism.

Biotechnological advances with applicability in potatoes for resistance against root-knot nematodes.

Potato production is negatively affected by root-knot nematodes (Meloidogyne spp.). There are no commercially available potato cultivars that are resistant to root-knot nematodes. To reduce the reliance on chemical controls, genetic engineering for nematode resistance in potato shows promise. Genetically modified potatoes that silence a parasitism gene or that express toxic protease inhibitors display reduced nematode infections. Modifying potato immune responses may also offer new opportunities for nematode resistance in potato. Plant defense elicitors, including those secreted by modified bacteria, enhanced resistance against root-knot nematodes in potato. The use of transgenic bacteria as delivery vehicles of defense-related molecules presents several possibilities for sophisticated nematode management and because this does not involve transgenic plants, it may garner greater public acceptance.

Nematode Genome Announcement: Draft Genome of Meloidogyne chitwoodi, an Economically Important Pest of Potato in the Pacific Northwest.

Meloidogyne chitwoodi is one of the most devastating pests of potato in the U.S. Pacific Northwest (PNW). Nematode-infected tubers develop external as well as internal defects, making the potato tubers unmarketable, and resulting in economic losses. Draft genome assemblies of three M. chitwoodi genotypes-race 1, race 2 and race 1 pathotype Roza-were generated using Illumina and PacBio Sequel RS II sequencing. The final assemblies consist of 30, 39, and 38 polished contigs for race 1, race 2 and race 1 pathotype Roza, respectively, with average N50 of 2.37 Mb and average assembled genome size of approximately 47.41 Mb. On average, 10,508 genes were annotated for each genome. Benchmarking universal single-copy ortholog (BUSCO) analysis indicated that 69.80% of the BUSCOs were complete whereas 68.80, 0.93, and 12.67% were single copy, duplicated, and fragmented, respectively. These highly contiguous genomes will enrich resources to study potato-nematode interactions and enhance breeding efforts to develop nematode-resistant potato varieties for the PNW.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Enhancing potato resistance against root-knot nematodes using a plant-defence elicitor delivered by bacteria.

The root-knot nematode Meloidogyne chitwoodi is a pest that affects potato production in the Pacific Northwest of the United States. Here, to develop new strategies against M. chitwoodi infection of potato, we engineered Bacillus subtilis to secrete the plant-defence elicitor peptide StPep1. Pre-treatment of potato roots with the bacteria secreting StPep1 substantially reduced root galling, indicating that a bacterial secretion of a plant elicitor is an effective strategy for plant protection.

Correction to: Transcriptome profiling of resistance response to Meloidogyne chitwoodi introgressed from wild species Solanum bulbocastanum into cultivated potato.

Following the publication of this article [1], the authors noted an error in Figure 11.

Transcriptome profiling of resistance response to Meloidogyne chitwoodi introgressed from wild species Solanum bulbocastanum into cultivated potato.

BACKGROUND: Meloidogyne chitwoodi commonly known as Columbia root-knot nematode or CRKN is one of the most devastating pests of potato in the Pacific Northwest of the United States of America. In addition to the roots, it infects potato tubers causing internal as well as external defects, thereby reducing the market value of the crop. Commercial potato varieties with CRKN resistance are currently unavailable. Race specific resistance to CRKN has been introgressed from the wild, diploid potato species Solanum bulbocastanum into the tetraploid advanced selection PA99N82-4 but there is limited knowledge about the nature of its resistance mechanism. In the present study, we performed histological and differential gene expression profiling to understand the mode of action of introgressed CRKN resistance in PA99N82-4 in comparison to the CRKN susceptible variety Russet Burbank. RESULTS: Histological studies revealed that the nematode juveniles successfully infect both resistant and susceptible root tissue by 48 h post inoculation, but the host resistance response restricts nematode feeding site formation in PA99N82-4. Differential gene expression analysis shows that 1268, 1261, 1102 and 2753 genes were up-regulated in PA99N82-4 at 48 h, 7 days, 14 days and 21 days post inoculation respectively, of which 61 genes were common across all the time points. These genes mapped to plant-pathogen interaction, plant hormonal signaling, antioxidant activity and cell wall re-enforcement pathways annotated for potato. CONCLUSION: The introgressed nematode resistance in PA99N82-4 is in the form of both pattern-triggered immune response and effector-triggered immune response, which is mediated by accumulation of reactive oxygen species and hypersensitive response (HR). Salicylic acid is playing a major role in the HR. Polyamines and suberin (a component of the Casperian strip in roots) also play an important role in mediating the resistance response. The present study provides the first ever comprehensive insights into transcriptional changes among M. chitwoodi resistant and susceptible potato genotypes after nematode inoculation. The knowledge generated in the present study has implications in breeding for CRKN resistance in potato.

Plant-parasitic nematode effectors - insights into their diversity and new tools for their identification.

Plant-parasitic nematodes (PPNs) are a large group of obligate biotrophic pathogens that secrete molecules, called effectors, involved in parasitism. The majority of work in molecular phytonematology has focused on the root-knot and cyst nematodes, which are both sedentary endoparasitic nematodes. More recently, inexpensive sequencing technology has facilitated effector searches in PPNs with different parasitic lifestyles. Work in different PPN species suggests that effectors are diverse, and selection pressure from plant hosts has contributed to the presence of large, expanded effector gene families. The identification of promoter elements/motifs preceding effector gene sequences suggests that promoter analysis can computationally predict new putative effectors. However, until a method of genetic transformation is available for PPNs, work on characterizing effectors will be hindered.

Loop-Mediated Isothermal Amplification for the Diagnostic Detection of Meloidogyne chitwoodi and M. fallax.

Meloidogyne chitwoodi is a root-knot nematode that parasitizes a broad range of plants. In the Pacific Northwest (PNW) of the United States, M. chitwoodi is a major potato pest. The nematodes infect roots and tubers; blemishes caused by the nematodes on the tubers significantly affect potato marketability. M. chitwoodi is a quarantine pathogen by many regulatory agencies, limiting potato trade opportunities when it is present. A loop-mediated isothermal amplification (LAMP) assay was developed to amplify the intergenic spacer (IGS2)-18S region of the ribosomal rDNA of M. chitwoodi. Using the LAMP assay, we could detect the presence of M. chitwoodi from infected Washington State soil samples. The LAMP primers showed specificity for DNA from M. chitwoodi and the closely related species M. fallax. There was no cross reaction of the LAMP primers with DNA from tropical nematodes M. incognita, M. arenaria, and M. javanica, or the Northern root-knot nematode M. hapla. The LAMP assays can be completed within 45 min, and they were 100 times more sensitive in nematode detection than conventional PCR. The LAMP assay will facilitate detection of potato nematodes M. chitwoodi and M. fallax. Knowledge of potato nematodes, particularly M. chitwoodi in PNW soils, will aid management decisions.

The root-knot nematode effector MiPFN3 disrupts plant actin filaments and promotes parasitism.

Root-knot nematodes secrete effectors that manipulate their host plant cells so that the nematode can successfully establish feeding sites and complete its lifecycle. The root-knot nematode feeding structures, their "giant cells," undergo extensive cytoskeletal remodeling. Previous cytological studies have shown the cytoplasmic actin within the feeding sites looks diffuse. In an effort to study root-knot nematode effectors that are involved in giant cell organogenesis, we have identified a nematode effector called MiPFN3 (Meloidogyne incognita Profilin 3). MiPFN3 is transcriptionally up-regulated in the juvenile stage of the nematode. In situ hybridization experiments showed that MiPFN3 transcribed in the nematode subventral glands, where it can be secreted by the nematode stylet into the plant. Moreover, Arabidopsis plants that heterologously expressed MiPFN3 were more susceptible to root-knot nematodes, indicating that MiPFN3 promotes nematode parasitism. Since profilin proteins can bind and sequester actin monomers, we investigated the function of MiPFN3 in relation to actin. Our results show that MiPFN3 suppressed the aberrant plant growth phenotype caused by the misexpression of reproductive actin (AtACT1) in transgenic plants. In addition, it disrupted actin polymerization in an in vitro assay, and it reduced the filamentous actin network when expressed in Arabidopsis protoplasts. Over a decade ago, cytological studies showed that the cytoplasmic actin within nematode giant cells looked fragmented. Here we provide the first evidence that the nematode is secreting an effector that has significant, direct effects on the plant's actin cytoskeleton.

W342F Mutation in CCaMK Enhances Its Affinity to Calmodulin But Compromises Its Role in Supporting Root Nodule Symbiosis in Medicago truncatula.

The calcium/calmodulin-dependent protein kinase (CCaMK) is regulated by free Ca2+ and Ca2+-loaded calmodulin. This dual binding is believed to be involved in its regulation and associated physiological functions, although direct experimental evidence for this is lacking. Here we document that site-directed mutations in the calmodulin-binding domain of CCaMK alters its binding capacity to calmodulin, providing an effective approach to study how calmodulin regulates CCaMK in terms of kinase activity and regulation of rhizobial symbiosis in Medicago truncatula. We observed that mutating the tryptophan at position 342 to phenylalanine (W342F) markedly increased the calmodulin-binding capability of the mutant. The mutant CCaMK underwent autophosphorylation and catalyzed substrate phosphorylation in the absence of calcium and calmodulin. When the mutant W342F was expressed in ccamk-1 roots, the transgenic roots exhibited an altered nodulation phenotype. These results indicate that altering the calmodulin-binding domain of CCaMK could generate a constitutively activated kinase with a negative role in the physiological function of CCaMK.

Heterodera schachtii Tyrosinase-like protein - a novel nematode effector modulating plant hormone homeostasis.

The beet cyst nematode Heterodera schachtii causes major yield losses in sugar beet. Understanding the interaction between H. schachtii and its host plant is important for developing a sustainable management system. Nematode effectors play a crucial role in initializing and sustaining successful parasitism. In our study, we identified a gene (Hs-Tyr) encoding a tyrosinase functional domain (PF00264). We describe Hs-Tyr as a novel nematode effector. Hs-Tyr is localized in the nematode esophageal gland. Up-regulation of its expression coincided with the parasitic developmental stages of the nematode. Silencing Hs-Tyr by RNA interference made the treated nematodes less virulent. When RNAi-treated nematodes succeeded in infecting the plant, developing females and their associated syncytial nurse cells were significantly smaller than in control plants. Ectopically expressing the Hs-Tyr effector in Arabidopsis increased plant susceptibility to H. schachtii, but not to the root-knot nematode Meloidogyne incognita. Interestingly, Hs-Tyr in the plant promoted plant growth and changed the root architecture. Additionally, the expression of Hs-Tyr in Arabidopsis caused changes in the homeostasis of several plant hormones especially auxin and the ethylene precursor aminocyclopropane-carboxylic acid.

Autophosphorylation of calcium/calmodulin-dependent protein kinase (CCaMK) at S343 or S344 generates an intramolecular interaction blocking the CaM-binding.

The Ca2+ and Ca2+/calmodulin-dependent protein kinase (CCaMK) is an important effector protein of Ca2+/calmodulin-mediated signaling, and in legumes, it is a critical regulator of plant-rhizobia and mycorrhizal symbioses. CCaMK contains a kinase domain, a calmodulin-binding/autoinhibitory domain and a visinin-like domain. Previous studies revealed the presence of 2 phosphorylation sites, S343 and S344, in the calmodulin-binding domain. Mutations at these sites affected the kinase activity and downstream rhizobium and mycorrhizal symbioses, which highlighted the importance of these residues in regulating protein activity. This addendum further clarifies the regulation of CCaMK by identifying an intramolecular interaction between residue(s) in the kinase domain and phosphorylation sites S343 and S344. This interaction turns off the substrate phosphorylation capacity of CCaMK.

Identification of Two Meloidogyne hapla Genes and an Investigation of Their Roles in the Plant-Nematode Interaction.

Root-knot nematodes are soil-borne pathogens that invade and establish feeding sites in plant roots. They have an extremely broad host range, including most vascular plants. During infection of a susceptible host, root-knot nematodes secrete molecules called effectors that help them establish an intimate interaction with the plant and, at the same time, allow them to evade or suppress plant immune responses. Despite the fact that Meloidogyne hapla is a significant pest on several food crops, no effectors have been characterized from this root-knot nematode species thus far. Using the published genome and proteome from M. hapla, we have identified and characterized two genes, MhTTL2 and Mh265. MhTTL2 encodes a predicted secreted protein containing a transthyretin-like protein domain. The expression of MhTTL2 was up-regulated during parasitic life stages of the nematode, and in situ hybridization showed that MhTTL2 was expressed in the amphids, suggesting it has a role in the nematode nervous system during parasitism. We also studied the gene Mh265. The Mh265 transcript was localized to the subventral esophageal glands. An upregulation in Mh265 expression coincided with the pre- and early-parasitic life stages of the nematode. When Mh265 was constitutively expressed in plants, it enhanced their susceptibility to nematodes. These transgenic plants were also compromised in flg22-induced callose deposition, suggesting the Mh265 is modulating plant basal immune responses.