Protein complexes from edible mushrooms as a sustainable potato protection against coleopteran pests.

Protein complexes from edible oyster mushrooms (Pleurotus sp.) composed of pleurotolysin A2 (PlyA2) and pleurotolysin B (PlyB) exert toxicity in feeding tests against Colorado potato beetle (CPB) larvae, acting through the interaction with insect-specific membrane sphingolipid. Here we present a new strategy for crop protection, based on in planta production of PlyA2/PlyB protein complexes, and we exemplify this strategy in construction of transgenic potato plants of cv Désirée. The transgenics in which PlyA2 was directed to the vacuole and PlyB to the endoplasmic reticulum are effectively protected from infestation by CPB larvae without impacting plant performance. These transgenic plants showed a pronounced effect on larval feeding rate, the larvae feeding on transgenic plants being on average five to six folds lighter than larvae feeding on controls. Further, only a fraction (11%-37%) of the larvae that fed on transgenic potato plants completed their life cycle and developed into adult beetles. Moreover, gene expression analysis of CPB larvae exposed to PlyA2/PlyB complexes revealed the response indicative of a general stress status of larvae and no evidence of possibility of developing resistance due to the functional inactivation of PlyA2/PlyB sphingolipid receptors.

The StPti5 ethylene response factor acts as a susceptibility factor by negatively regulating the potato immune response to pathogens.

Ethylene response factors (ERFs) have been associated with biotic stress in Arabidopsis, while their function in non-model plants is still poorly understood. Here we investigated the role of potato ERF StPti5 in plant immunity. We show that StPti5 acts as a susceptibility factor. It negatively regulates potato immunity against potato virus Y and Ralstonia solanacearum, pathogens with completely different modes of action, and thereby has a different role than its orthologue in tomato. Remarkably, StPti5 is destabilised in healthy plants via the autophagy pathway and accumulates exclusively in the nucleus upon infection. We demonstrate that StEIN3 and StEIL1 directly bind the StPti5 promoter and activate its expression, while synergistic activity of the ethylene and salicylic acid pathways is required for regulated StPti expression. To gain further insight into the mode of StPti5 action in attenuating potato defence responses, we investigated transcriptional changes in salicylic acid deficient potato lines with silenced StPti5 expression. We show that StPti5 regulates the expression of other ERFs and downregulates the ubiquitin-proteasome pathway as well as several proteases involved in directed proteolysis. This study adds a novel element to the complex puzzle of immune regulation, by deciphering a two-level regulation of ERF transcription factor activity in response to pathogens.

A mini-TGA protein modulates gene expression through heterogeneous association with transcription factors.

TGA (TGACG-binding) transcription factors, which bind their target DNA through a conserved basic region leucine zipper (bZIP) domain, are vital regulators of gene expression in salicylic acid (SA)-mediated plant immunity. Here, we investigated the role of StTGA2.1, a potato (Solanum tuberosum) TGA lacking the full bZIP, which we named a mini-TGA. Such truncated proteins have been widely assigned as loss-of-function mutants. We, however, confirmed that StTGA2.1 overexpression compensates for SA-deficiency, indicating a distinct mechanism of action compared with model plant species. To understand the underlying mechanisms, we showed that StTGA2.1 can physically interact with StTGA2.2 and StTGA2.3, while its interaction with DNA was not detected. We investigated the changes in transcriptional regulation due to StTGA2.1 overexpression, identifying direct and indirect target genes. Using in planta transactivation assays, we confirmed that StTGA2.1 interacts with StTGA2.3 to activate StPRX07, a member of class III peroxidases (StPRX), which are known to play role in immune response. Finally, via structural modeling and molecular dynamics simulations, we hypothesized that the compact molecular architecture of StTGA2.1 distorts DNA conformation upon heterodimer binding to enable transcriptional activation. This study demonstrates how protein truncation can lead to distinct functions and that such events should be studied carefully in other protein families.

Chloroplast redox state changes mark cell-to-cell signaling in the hypersensitive response.

Hypersensitive response (HR)-conferred resistance is associated with induction of programmed cell death and pathogen spread restriction in its proximity. The exact role of chloroplastic reactive oxygen species and its link with salicylic acid (SA) signaling in HR remain unexplained. To unravel this, we performed a detailed spatiotemporal analysis of chloroplast redox response in palisade mesophyll and upper epidermis to potato virus Y (PVY) infection in a resistant potato genotype and its transgenic counterpart with impaired SA accumulation and compromised resistance. Besides the cells close to the cell death zone, we detected individual cells with oxidized chloroplasts further from the cell death zone. These are rare in SA-deficient plants, suggesting their role in signaling for resistance. We confirmed that chloroplast redox changes play important roles in signaling for resistance, as blocking chloroplast redox changes affected spatial responses at the transcriptional level. Through spatiotemporal study of stromule induction after PVY infection, we show that stromules are induced by cell death and also as a response to PVY multiplication at the front of infection. Overall induction of stromules is attenuated in SA-deficient plants.

Organelles and phytohormones: a network of interactions in plant stress responses.

Phytohormones are major signaling components that contribute to nearly all aspects of plant life. They constitute an interconnected communication network to fine-tune growth and development in response to the ever-changing environment. To this end, they have to coordinate with other signaling components, such as reactive oxygen species and calcium signals. On the one hand, the two endosymbiotic organelles, plastids and mitochondria, control various aspects of phytohormone signaling and harbor important steps of hormone precursor biosynthesis. On the other hand, phytohormones have feedback actions on organellar functions. In addition, organelles and phytohormones often act in parallel in a coordinated matter to regulate cellular functions. Therefore, linking organelle functions with increasing knowledge of phytohormone biosynthesis, perception, and signaling will reveal new aspects of plant stress tolerance. In this review, we highlight recent work on organelle-phytohormone interactions focusing on the major stress-related hormones abscisic acid, jasmonates, salicylic acid, and ethylene.

Intertwined Roles of Reactive Oxygen Species and Salicylic Acid Signaling Are Crucial for the Plant Response to Biotic Stress.

One of the earliest hallmarks of plant immune response is production of reactive oxygen species (ROS) in different subcellular compartments, which regulate plant immunity. A suitable equilibrium, which is crucial to prevent ROS overaccumulation leading to oxidative stress, is maintained by salicylic acid (SA), a chief regulator of ROS. However, ROS not only act downstream of SA signaling, but are also proposed to be a central component of a self-amplifying loop that regulates SA signaling as well as the interaction balance between different phytohormones. The exact role of this crosstalk, the position where SA interferes with ROS signaling and ROS interferes with SA signaling and the outcome of this regulation, depend on the origin of ROS but also on the pathosystem. The precise spatiotemporal regulation of organelle-specific ROS and SA levels determine the effectiveness of pathogen arrest and is therefore crucial for a successful immune response. However, the regulatory interplay behind still remains poorly understood, as up until now, the role of organelle-specific ROS and SA in hypersensitive response (HR)-conferred resistance has mostly been studied by altering the level of a single component. In order to address these aspects, a sophisticated combination of research methods for monitoring the spatiotemporal dynamics of key players and transcriptional activity in plants is needed and will most probably consist of biosensors and precision transcriptomics.

Precision transcriptomics of viral foci reveals the spatial regulation of immune-signaling genes and identifies RBOHD as an important player in the incompatible interaction between potato virus Y and potato.

Whereas the activation of resistance (R) proteins has been intensively studied, the downstream signaling mechanisms leading to the restriction of the pathogen remain mostly unknown. We studied the immunity network response conditioned by the potato Ny-1 gene against potato virus Y. We analyzed the processes in the cell death zone and surrounding tissue on the biochemical and gene expression levels in order to reveal the spatiotemporal regulation of the immune response. We show that the transcriptional response in the cell death zone and surrounding tissue is dependent on salicylic acid (SA). For some genes the spatiotemporal regulation is completely lost in the SA-deficient line, whereas other genes show a different response, indicating multiple connections between hormonal signaling modules. The induction of NADPH oxidase RBOHD expression occurs specifically on the lesion border during the resistance response. In plants with silenced RBOHD, the functionality of the resistance response is perturbed and the spread of the virus is not arrested at the site of infection. RBOHD is required for the spatial accumulation of SA, and conversely RBOHD is under the transcriptional regulation of SA. Using spatially resolved RNA-seq, we also identified spatial regulation of an UDP-glucosyltransferase, another component in feedback activation of SA biosynthesis, thus deciphering a novel aspect of resistance signaling.

Studying Cell Death Initiation Using a Digital Microscope.

Hypersensitive response (HR)-conferred resistance is an effective defense response that can be determined by the N resistance genes. HR is manifested as the formation of cell death zones on inoculated leaves. Here, a protocol for studying the rate of cell death initiation by imaging inoculated leaves in the time between the cell death initiation and the cell death appearance using a digital microscope is presented. The digital microscope enables a continuous imaging process in desired intervals, which allows an accurate determination of cell death initiation rate up to minutes exactly, as opposed to hours in traditional methods. Imaging with the digital microscope is also independent of light and can therefore be used during day and night without disturbing the circadian rhythm of the plant. Different pathosystems resulting in programmed cell death development could be studied using this protocol with minor modifications. Overall, the protocol thus allows simple, accurate, and inexpensive identification of cell death initiation rate.

Candidate pathogenicity factor/effector proteins of 'Candidatus Phytoplasma solani' modulate plant carbohydrate metabolism, accelerate the ascorbate-glutathione cycle, and induce autophagosomes.

The pathogenicity of intracellular plant pathogenic bacteria is associated with the action of pathogenicity factors/effectors, but their physiological roles for most phytoplasma species, including 'Candidiatus Phytoplasma solani' are unknown. Six putative pathogenicity factors/effectors from six different strains of 'Ca. P. solani' were selected by bioinformatic analysis. The way in which they manipulate the host cellular machinery was elucidated by analyzing Nicotiana benthamiana leaves after Agrobacterium-mediated transient transformation with the pathogenicity factor/effector constructs using confocal microscopy, pull-down, and co-immunoprecipitation, and enzyme assays. Candidate pathogenicity factors/effectors were shown to modulate plant carbohydrate metabolism and the ascorbate-glutathione cycle and to induce autophagosomes. PoStoSP06, PoStoSP13, and PoStoSP28 were localized in the nucleus and cytosol. The most active effector in the processes studied was PoStoSP06. PoStoSP18 was associated with an increase in phosphoglucomutase activity, whereas PoStoSP28, previously annotated as an antigenic membrane protein StAMP, specifically interacted with phosphoglucomutase. PoStoSP04 induced only the ascorbate-glutathione cycle along with other pathogenicity factors/effectors. Candidate pathogenicity factors/effectors were involved in reprogramming host carbohydrate metabolism in favor of phytoplasma own growth and infection. They were specifically associated with three distinct metabolic pathways leading to fructose-6-phosphate as an input substrate for glycolysis. The possible significance of autophagosome induction by PoStoSP28 is discussed.

CRISPR/Cas-mediated plant genome editing: outstanding challenges a decade after implementation.

The discovery of the CRISPR/Cas genome-editing system has revolutionized our understanding of the plant genome. CRISPR/Cas has been used for over a decade to modify plant genomes for the study of specific genes and biosynthetic pathways as well as to speed up breeding in many plant species, including both model and non-model crops. Although the CRISPR/Cas system is very efficient for genome editing, many bottlenecks and challenges slow down further improvement and applications. In this review we discuss the challenges that can occur during tissue culture, transformation, regeneration, and mutant detection. We also review the opportunities provided by new CRISPR platforms and specific applications related to gene regulation, abiotic and biotic stress response improvement, and de novo domestication of plants.

Analysis of Virus Spread Around the Cell Death Zone at Spatiotemporal Resolution Using Confocal Microscopy.

The role of programmed cell death (PCD) in hypersensitive response (HR)-conferred resistance depends on the type of host-pathogen interaction and therefore has to be studied for each individual pathosystem. Here we present and explain the protocol for studying the role of PCD in HR-conferred resistance in potato plants in the interaction with the viral pathogen. As an experimental system, we use genotype Rywal, where the virus spread is restricted and HR PCD develops 3 days post potato virus Y (PVY) inoculation. As a control of virus multiplication and spread, we include its transgenic counterpart impaired in salicylic acid (SA) accumulation (NahG-Rywal), in which the HR-PCD occurs but the spread of the virus is not restricted. To follow the occurrence of virus-infected cells and/or virus multiplication outside the cell death zone, we use GFP-tagged PVY (PVY-N605(123)-GFP) which can be monitored by confocal microscopy. Any other plant-pathogen system which results in PCD development could be studied using a modified version of this protocol.

Plant X-tender Toolbox for the Assembly and Expression of Multiple Transcriptional Units in Plants.

Plant synthetic biology requires the design of plant expression vectors with multiple transcriptional units, which can be challenging. Here we describe the use of Plant X-tender toolbox complemented with a plant grammar implemented in GenoCAD for design, cloning and delivery of several transcriptional units into the plant genome. Plant X-tender toolbox consists of a set of plant expression vectors and the protocols for the most efficient cloning of multiple transcriptional units into this novel vector set. Together with the plant grammar implemented in GenoCAD, the presented strategy allows the users to quickly design genetic modules and assemble them into Plant X-tender expression vectors for in planta functional studies or synthetic biology applications.

CRISPR/Cas9-mediated fine-tuning of miRNA expression in tetraploid potato.

MicroRNAs (miRNAs) are small noncoding RNAs, which modulate the abundance and spatiotemporal accumulation of target mRNAs at transcriptional and post-transcriptional levels and through that play important roles in several biological processes in plants. Here we show that in polyploid species, CRISPR/Cas9 system can be used for fine-tuning of miRNA expression, which can have broader range of applications compared to knock-out mutants. We established the complete pipeline for CRISPR-Cas9-mediated modulation of miRNA expression in potato. It consists of (1) design and assembly of dual sgRNA CRISPR/Cas9 constructs, (2) transient transfection of protoplasts following fast and efficient screening by high resolution melting analysis to select functional sgRNAs, and (3) stable transformation of potato explants with functional sgRNAs and selection of regenerated transgenic lines with desired mutations and desired miRNA abundance based on sequencing and RT-qPCR. We show that miRNA-editing using dual sgRNA approach results in different types of mutations among transgenic lines but also in different alleles of the same plant, which are target site-dependent. The most frequent were short deletions, but we also detected 1-nt insertions (T or G), deletions between two sgRNAs and larger deletions. miRNA abundance correlates with the frequency and type of introduced mutations, as more extensive mutations in more alleles result in lower miRNA abundance. Interestingly, some mutated loci can generate alternative miRNAs, now novel targets were however predicted for those. In all transgenic lines with Cas9 expression, we detected mutations, suggesting high efficiency of Cas9-editing. We confirmed the miRNA-editing efficiency of our optimised approach in two different potato genotypes and three different loci.

Biosensors: A Sneak Peek into Plant Cell's Immunity.

Biosensors are indispensable tools to understand a plant's immunity as its spatiotemporal dimension is key in withstanding complex plant immune signaling. The diversity of genetically encoded biosensors in plants is expanding, covering new analytes with ever higher sensitivity and robustness, but their assortment is limited in some respects, such as their use in following biotic stress response, employing more than one biosensor in the same chassis, and their implementation into crops. In this review, we focused on the available biosensors that encompass these aspects. We show that in vivo imaging of calcium and reactive oxygen species is satisfactorily covered with the available genetically encoded biosensors, while on the other hand they are still underrepresented when it comes to imaging of the main three hormonal players in the immune response: salicylic acid, ethylene and jasmonic acid. Following more than one analyte in the same chassis, upon one or more conditions, has so far been possible by using the most advanced genetically encoded biosensors in plants which allow the monitoring of calcium and the two main hormonal pathways involved in plant development, auxin and cytokinin. These kinds of biosensor are also the most evolved in crops. In the last section, we examine the challenges in the use of biosensors and demonstrate some strategies to overcome them.

Gene Editing in Potato Using CRISPR-Cas9 Technology.

Genome editing in the cultivated potato (Solanum tuberosum), a vegetatively propagated and highly heterozygous species, constitutes a promising trail to directly improve traits into elite cultivars. With the recent and successful development of the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system in eukaryotic cells, the plant science community has gained access to a powerful, inexpensive, and easy-to-use toolbox to target and inactivate/modify specific genes. The specificity and versatility of the CRISPR-Cas9 system rely on a variable 20 bp spacer sequence at the 5' end of a single-guide RNA (sgRNA), which directs the SpCas9 (Streptococcus pyogenes) nuclease to cut the target DNA at a precise locus with no or low off-target events. Using this system, we and other teams were able to knock out specific genes in potato through the error-prone non-homologous end-joining (NHEJ) DNA repair mechanism. In this chapter, we describe strategies to design and clone spacer sequences into CRISPR-SpCas9 plasmids. We show how these constructs can be used for Agrobacterium-mediated stable transformation or transient transfection of protoplasts, and we describe the optimization of these two delivery methods, as well as of the plant regeneration processes. Finally, the molecular screening and characterization of edited potato plants are also described, mainly relying on PCR-based methods such as high-resolution melt (HRM) analysis.

Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants.

Cloning multiple DNA fragments for delivery of several genes of interest into the plant genome is one of the main technological challenges in plant synthetic biology. Despite several modular assembly methods developed in recent years, the plant biotechnology community has not widely adopted them yet, probably due to the lack of appropriate vectors and software tools. Here we present Plant X-tender, an extension of the highly efficient, scar-free and sequence-independent multigene assembly strategy AssemblX, based on overlap-depended cloning methods and rare-cutting restriction enzymes. Plant X-tender consists of a set of plant expression vectors and the protocols for most efficient cloning into the novel vector set needed for plant expression and thus introduces advantages of AssemblX into plant synthetic biology. The novel vector set covers different backbones and selection markers to allow full design flexibility. We have included ccdB counterselection, thereby allowing the transfer of multigene constructs into the novel vector set in a straightforward and highly efficient way. Vectors are available as empty backbones and are fully flexible regarding the orientation of expression cassettes and addition of linkers between them, if required. We optimised the assembly and subcloning protocol by testing different scar-less assembly approaches: the noncommercial SLiCE and TAR methods and the commercial Gibson assembly and NEBuilder HiFi DNA assembly kits. Plant X-tender was applicable even in combination with low efficient homemade chemically competent or electrocompetent Escherichia coli. We have further validated the developed procedure for plant protein expression by cloning two cassettes into the newly developed vectors and subsequently transferred them to Nicotiana benthamiana in a transient expression setup. Thereby we show that multigene constructs can be delivered into plant cells in a streamlined and highly efficient way. Our results will support faster introduction of synthetic biology into plant science.

Cell Death Is Not Sufficient for the Restriction of Potato Virus Y Spread in Hypersensitive Response-Conferred Resistance in Potato.

Hypersensitive response (HR)-conferred resistance to viral infection restricts the virus spread and is accompanied by the induction of cell death, manifested as the formation of necrotic lesions. While it is known that salicylic acid is the key component in the orchestration of the events restricting viral spread in HR, the exact function of the cell death in resistance is still unknown. We show that potato virus Y (PVY) can be detected outside the cell death zone in Ny-1-mediated HR in potato plants (cv. Rywal), observed as individual infected cells or small clusters of infected cells outside the cell death zone. By exploiting the features of temperature dependent Ny-1-mediated resistance, we confirmed that the cells at the border of the cell death zone are alive and harbor viable PVY that is able to reinitiate infection. To get additional insights into this phenomenon we further studied the dynamics of both cell death zone expansion and occurrence of viral infected cell islands outside it. We compared the response of Rywal plants to their transgenic counterparts, impaired in SA accumulation (NahG-Rywal), where the lesions occur but the spread of the virus is not restricted. We show that the virus is detected outside the cell death zone in all lesion developmental stages of HR lesions. We also measured the dynamics of lesions expansion in both genotypes. We show that while rapid lesion expansion is observed in SA-depleted plants, virus spread is even faster. On the other hand the majority of analyzed lesions slowly expand also in HR-conferred resistance opening the possibility that the infected cells are eventually engulfed by cell death zone. Taken altogether, we suggest that the HR cell death is separated from the resistance mechanisms which lead to PVY restriction in Ny-1 genetic background. We propose that HR should be regarded as a process where the dynamics of events is crucial for effectiveness of viral arrest albeit the exact mechanism conferring this resistance remains unknown.