Identification of the potato (Solanum tuberosum L.) P-type ATPase gene family and investigating the role of PHA2 in response to Pep13.

P-type ATPase family members play important roles in plant growth and development and are involved in plant resistance to various biotic and abiotic factors. Extensive studies have been conducted on the P-type ATPase gene families in Arabidopsis thaliana and rice but our understanding in potato remains relatively limited. Therefore, this study aimed to screen and analyze 48 P-type ATPase genes from the potato (Solanum tuberosum L.) genome database at the genome-wide level. Potato P-type ATPase genes were categorized into five subgroups based on the phylogenetic classification of the reported species. Additionally, several bioinformatic analyses, including gene structure analysis, chromosomal position analysis, and identification of conserved motifs and promoter cis-acting elements, were performed. Interestingly, the plasma membrane H+-ATPase (PM H+-ATPase) genes of one of the P3 subgroups showed differential expression in different tissues of potato. Specifically, PHA2, PHA3, and PHA7 were highly expressed in the roots, whereas PHA8 was expressed in potatoes only under stress. Furthermore, the small peptide Pep13 inhibited the expression of PHA1, PHA2, PHA3, and PHA7 in potato roots. Transgenic plants heterologously overexpressing PHA2 displayed a growth phenotype sensitive to Pep13 compared with wild-type plants. Further analysis revealed that reducing potato PM H+-ATPase enzyme activity enhanced resistance to Pep13, indicating the involvement of PM H+-ATPase in the physiological process of potato late blight and the enhancement of plant disease resistance. This study confirms the critical role of potato PHA2 in resistance to Pep13.

Spotting priming-active compounds using parsley cell cultures in microtiter plates.

BACKGROUND: Conventional crop protection has major drawbacks, such as developing pest and pathogen insensitivity to pesticides and low environmental compatibility. Therefore, alternative crop protection strategies are needed. One promising approach treats crops with chemical compounds that induce the primed state of enhanced defense. However, identifying priming compounds is often tedious as it requires offline sampling and analysis. High throughput screening methods for the analysis of priming-active compounds have great potential to simplify the search for such compounds. One established method to identify priming makes use of parsley cell cultures. This method relies on measurement of fluorescence of furanocoumarins in the final sample. This study demonstrates for the first time the online measurement of furanocoumarins in microtiter plates. As not all plants produce fluorescence molecules as immune response, a signal, which is not restricted to a specific plant is required, to extend online screening methods to other plant cell cultures. It was shown that the breathing activity of primed parsley cell cultures increases, compared to unprimed parsley cell cultures. The breathing activity can by monitored online. Therefore, online identification of priming-inducing compounds by recording breathing activity represents a promising, straight-forward and highly informative approach. However, so far breathing has been recorded in shake flasks which suffer from low throughput. For industrial application we here report a high-throughput, online identification method for identifying priming-inducing chemistry. RESULTS: This study describes the development of a high-throughput screening system that enables identifying and analyzing the impact of defense priming-inducing compounds in microtiter plates. This screening system relies on the breathing activity of parsley cell cultures. The validity of measuring the breathing activity in microtiter plates to drawing conclusions regarding priming-inducing activity was demonstrated. Furthermore, for the first time, the fluorescence of the priming-active reference compound salicylic acid and of furanocoumarins were simultaneously monitored online. Dose and time studies with salicylic acid-treated parsley cell suspensions revealed a wide range of possible addition times and concentrations that cause priming. The online fluorescence measuring method was further confirmed with three additional compounds with known priming-causing activity. CONCLUSIONS: Determining the OTR, fluorescence of the priming-active chemical compound SA and of furanocoumarins in parsley suspension cultures in MTPs by online measurement is a powerful and high-throughput tool to study possible priming compounds. It allows an in-depth screening for priming compounds and a better understanding of the priming process induced by a given substance. Evaluation of priming phenomena via OTR should also be applicable to cell suspensions of other plant species and varieties and allow screening for priming-inducing chemical compounds in intact plants. These online fluorescence methods to measure the breathing activity, furanocoumarin and SA have the potential to accelerate the search for new priming compounds and promote priming as a promising, eco-friendly crop protection strategy.

Elicitation of Roots and AC-DC with PEP-13 Peptide Shows Differential Defense Responses in Multi-Omics.

The root extracellular trap (RET) has emerged as a specialized compartment consisting of root AC-DC and mucilage. However, the RET's contribution to plant defense is still poorly understood. While the roles of polysaccharides and glycoproteins secreted by root AC-DC have started to be elucidated, how the low-molecular-weight exudates of the RET contribute to root defense is poorly known. In order to better understand the RET and its defense response, the transcriptomes, proteomes and metabolomes of roots, root AC-DC and mucilage of soybean (Glycine max (L.) Merr, var. Castetis) upon elicitation with the peptide PEP-13 were investigated. This peptide is derived from the pathogenic oomycete Phytophthora sojae. In this study, the root and the RET responses to elicitation were dissected and sequenced using transcriptional, proteomic and metabolomic approaches. The major finding is increased synthesis and secretion of specialized metabolites upon induced defense activation following PEP-13 peptide elicitation. This study provides novel findings related to the pivotal role of the root extracellular trap in root defense.

Recognition of Pep-13/25 MAMPs of Phytophthora localizes to an RLK locus in Solanum microdontum.

Pattern-triggered immunity (PTI) in plants is mediated by cell surface-localized pattern recognition receptors (PRRs) upon perception of microbe-associated molecular pattern (MAMPs). MAMPs are conserved molecules across microbe species, or even kingdoms, and PRRs can confer broad-spectrum disease resistance. Pep-13/25 are well-characterized MAMPs in Phytophthora species, which are renowned devastating oomycete pathogens of potato and other plants, and for which genetic resistance is highly wanted. Pep-13/25 are derived from a 42 kDa transglutaminase GP42, but their cognate PRR has remained unknown. Here, we genetically mapped a novel surface immune receptor that recognizes Pep-25. By using effectoromics screening, we characterized the recognition spectrum of Pep-13/25 in diverse Solanaceae species. Response to Pep-13/25 was predominantly found in potato and related wild tuber-bearing Solanum species. Bulk-segregant RNA sequencing (BSR-Seq) and genetic mapping the response to Pep-25 led to a 0.081 cM region on the top of chromosome 3 in the wild potato species Solanum microdontum subsp. gigantophyllum. Some BAC clones in this region were isolated and sequenced, and we found the Pep-25 receptor locates in a complex receptor-like kinase (RLK) locus. This study is an important step toward the identification of the Pep-13/25 receptor, which can potentially lead to broad application in potato and various other hosts of Phytophthora species.

New Insights into Plant Extracellular DNA. A Study in Soybean Root Extracellular Trap.

exDNA is found in various organisms, including plants. However, plant exDNA has thus far received little attention related to its origin and role in the RET (root extracellular trap). In this study, we performed the first high-throughput genomic sequencing of plant exDNA from a Fabaceae with worldwide interest: soybean (Glycine max (L.) Merr.). The origin of this exDNA was first investigated in control condition, and the results show high-coverage on organelles (mitochondria/plastid) DNA relative to nuclear DNA, as well as a mix of coding and non-coding sequences. In the second part of this study, we investigated if exDNA release was modified during an elicitation with PEP-13 (a peptide elicitor from oomycete genus Phytophthora). Our results show that treatment of roots with PEP-13 does not affect the composition of exDNA.

A phosphoinositide 5-phosphatase from Solanum tuberosum is activated by PAMP-treatment and may antagonize phosphatidylinositol 4,5-bisphosphate at Phytophthora infestans infection sites.

Potato (Solanum tuberosum) plants susceptible to late blight disease caused by the oomycete Phytophthora infestans display enhanced resistance upon infiltration with the pathogen-associated molecular pattern (PAMP), Pep-13. Here, we characterize a potato gene similar to Arabidopsis 5-phosphatases which was identified in transcript arrays performed to identify Pep-13 regulated genes, and termed StIPP. Recombinant StIPP protein specifically dephosphorylated the D5-position of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2 ) in vitro. Other phosphoinositides or soluble inositolpolyphosphates were not converted. When transiently expressed in tobacco (Nicotiana tabacum) pollen tubes, a StIPP-YFP fusion localized to the subapical plasma membrane and antagonized PtdIns(4,5)P2 -dependent effects on cell morphology, indicating in vivo functionality. Phytophthora infestans-infection of N. benthamiana leaf epidermis cells resulted in relocalization of StIPP-GFP from the plasma membrane to the extra-haustorial membrane (EHM). Colocalizion with the effector protein RFP-AvrBlb2 at infection sites is consistent with a role of StIPP in the plant-oomycete interaction. Correlation analysis of fluorescence distributions of StIPP-GFP and biosensors for PtdIns(4,5)P2 or phosphatidylinositol 4-phosphate (PtdIns4P) indicate StIPP activity predominantly at the EHM. In Arabidopsis protoplasts, expression of StIPP resulted in the stabilization of the PAMP receptor, FLAGELLIN-SENSITIVE 2, indicating that StIPP may act as a PAMP-induced and localized antagonist of PtdIns(4,5)P2 -dependent processes during plant immunity.