Effect of Vicia sativa L. on Motility, Mortality and Expression Levels of hsp Genes in J2 Stage of Meloidogyne hapla

Plants and nematodes used for research: Common vetch Vicia sativa L.) seeds were obtained from Plant Breeding DANKO, based in Szelejewo, Poland, and tomato Solanum lycopersicum L., cv. Betalux) seeds were obtained from from PNOS, based in Ożarów Mazowiecki, Poland. The seeds of Ina cultivar with the content of 7.9 mg% dry weight of cyanogenic compounds and the Jaga cultivar with the content of 3.5 mg% dry weight were used for the tests (COBORU, 2022).

M. hapla population was collected from a carrot Daucus carota L.) field and morphologically and genetically identified in accordance with Karssen’s (1999) and Petersen and Vrain’s (1996) diagnostic protocols, respectively. The population was maintained on tomato plants grown in a greenhouse under conditions of 20 ± 1°C and day and night length (16/8). M. hapla J2 juveniles were obtained from the eggs using the modified Baermann method described by Rodríguez-Kábana and Pope (1981). Only J2s that had left their eggshells within 24 hours prior to the start of the experiment were used in the study.

Exposure of J2 stage to V. sativa seed diffusates: Only the diffusate from non-sterilized vetch seeds of the Ina cultivar in water was used in the study. The Ina cultivar is characterized by more than twice the content of cyanogenic compounds per gram of dry seeds compared to the Jaga cultivar. Three separate experiments were performed. They were carried out at a temperature of 21 ± 1°C. Each experiment was repeated three times. 200 specimens of the J2 stage of M. hapla were used for each run. In the first experiment, the nematodes were placed in Petri dishes in a diffusate for 24 hours. Each of the three Petri dishes with the diffusate contained 200 specimens of the J2 stage specimens. After this time, the immobile (paralysed) nematodes were transferred to Eppendorf tubes and immediately preserved with phenosol (RNA preservative reagent by A&A Biotechnology RNA) and frozen at -80°C, until obtaining the isolation of total RNA. In the second experiment, nematodes (200 specimens x 3 repetitions) were placed in a common vetch seed diffusate for 24 h and then transferred to water for 24 h. In this experiment, 24 hours after being transferred from the diffusate to water, almost all the nematodes regained the ability to move (approximately 8% of the specimens remained stationary in each run of the experiment). The specimens were then preserved with phenosol and immediately frozen at -80°C until RNA isolation. In the third experiment, J2 specimens (200 specimens × 3 repetitions) were left in the water for 24 hours, the control sample. The control sample was then preserved and frozen at -80°C.

RNA extraction: The isolation of total RNA from the three experiments described above was performed by the modified phenol-chloroform method with the use of the A&A Biotechnology kit Chomczynski and Sacchi, 1987. RNA extraction was performed in the pre-PCR room using disposable DNase- and RNase-free pipette tips with filters and test tubes. All stages of the experiment were performed at a temperature of approx. 4°C. The obtained RNA was stored at -80°C until further analysis. The quantity and quality of the obtained RNA was determined using the NanoDrop 1000 Spectrophotometer vs. 3.7 (Thermo Scientific). RNA integrity was assessed by electrophoresis using the RiboRuler High Range RNA Ladder (Thermo Scientific).

cDNA synthesis: DNase digestion of the obtained RNA and first-strand cDNA synthesis were performed with the iScript ™ gDNA Clear cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) according to the procedure described by the manufacturer. The DNAse reaction mixture, in the amount of 0.5 μl and 1.5 μl of DNAse buffer for each 14 μl of RNA, was used to treat the residual DNA in the isolated RNA at a concentration of 10 ng / μl. The mixture thus prepared was incubated for 5 min at 25°C. It was then incubated for 5 min at 75°C to inactivate DNase and transferred to ice. The RNA prepared in this manner was used for the synthesis of cDNA. However, if longer storage was necessary, the RNA sample was placed at -80°C.

For cDNA synthesis, we used 16 μl of DNase treated RNA and 4 μl of the reaction mixture containing i.a. iScript reverse transcriptase enzyme, RNAase inhibitor, primers oligo (dT), free nucleotides (dNTPs), magnesium chloride (MgCl₂ and polymerase stabilizing substances. The synthesis was carried out in the following steps: priming – 5 min at 25°C; reverse transcription (cDNA synthesis) – 20 min at 46°C; inactivation of the enzyme reverse transcriptase – 1 min at 95°C. The obtained first strand of cDNA was used directly in a quantitative polymerase chain reaction (qPCR) analysis or stored at -20°C.

Primer design: The primers for the tested genes Mh–hsp90, Mh–hsp1, Mh–hsp60, Mh–hsp43 and Mh–hsp12.3 used in the qPCR reaction were designed using the PRIMER3 vs. 0.4.0 program Untergasser et al., 2012. The Merck company performed the synthesis of the designed primers. The reference malate dehydrogenase Mdh gene and primers for amplifying a fragment of this gene (forward 5´–GAAAGCCAGGGATGACAC–3´, reverse 5´–AGAAAAGCATTGGGACAG–3´) were selected on the basis of research by Wu et al. (2019a). The Mdh gene has been shown to be one of the most suitable reference genes for gene expression studies in M. hapla.

qPCR reaction: We analyzed the expression of five hsp genes Mh–hsp90, Mh–hsp1, Mh–hsp60, Mh–hsp43, and Mh–hsp12.3). Expression of the 2^ΔΔ Ct method Livak and Schmittgen, 2001 was used in calculating the relative ratio, but instead of the value being 2, the correct amplification efficiency was used. We used a noise-resistant iterative nonlinear regression algorithm (qPCR miner) to determine the efficiency of the PCR reaction Zhao and Fernald, 2005. The expression levels are indicated as the fold-change normalized to the control (untreated diffusate), normalized to the value of 1.

qPCRs were performed on the 7500 qPCR system (Applied Biosystems, Waltham, MA, USA). Reactions were carried out using SsoAdvanced Universal SYBR Green Supermix kit (Bio-Rad), while cycle threshold (Ct) estimates were obtained using the relative quantification module in the software package. PCR reactions were performed in a final volume of 20 μl containing 1.5 μl of the cDNA sample, 3.0 μl of the primer mix (5 micromoles of each primer), 10 μl of the 2x SsoAdvanced Universal SYBR Green Supermix, and 5.5 μl of H₂O. The assay included a no template control and each of the test cDNAs from three biological replications. According to the instructions for this kit, after 30 sec. at 95°C, the cycling conditions were as follows: 40 cycles at 95°C for 15 sec. and 59°C for 60 sec. To validate the specificity of amplification, a post-amplification melt-curve analysis was performed. Amplicons were first denatured at 95°C for 60 sec. and then cooled to 72°C, and the temperature was then gradually raised to 95°C. Fluorescence data were recorded continuously during this period, and analyzed subsequently using the Tm calling module in the SDS Software v1.4 software.

Statistical analysis: Hsp gene expression values were expressed as the mean fold difference. Statistically significant differences between treated and control samples (p≤0.01 based on t-Student test, using an online tool “Do my qPCR calculation”) are shown Tournayre et al., 2019.

The results of observation of the motility and mortality of J2 stage of the northern root–knot nematode, as well the expression profile of selected hsp genes, showed that common vetch seeds affect the J2 stage of M. hapla, which is important for the population dynamics of this root-knot nematode species.

When exposed to diffusates of common vetch seeds, invasive J2 were found to lose their motility, which was probably due to the occurrence of oxidative stress caused by the action of HCN present in the diffusate. It has been proven that HCN is toxic to nematodes, and at a relevant concentration, it causes paralysis and even death of specimens of the model organism, the free-living nematode Caenorhabdidtis elegans Gallagher and Manoil, 2001; Curto et al., 2012; Ayuda-Durán et al., 2020. The motile behaviour of nematodes determines their ability as a parasitic species to invade the root tissues of the host plant and their further development within Gray and Lissmann, 1964. The paralysis and mortality of the invasive J2 stages immersed in common vetch seed diffusates indicate that, even at the stage of seeds in the soil, plants may have a limiting effect on invasive nematodes that are potentially able to infect them. The presence of cyanogenic compounds and the release of HCN into the soil may explain the slight increase in the nematode population observed in the natural environment where V. sativa Dobosz and Krawczyk, 2019; Mosjidis et al., 1994 and other species of the genus Vicia are grown (Moneim and Bellar, 1993). Similarly, the poor suitability of Sorghum spp. and its inter-specific hybrids as plant hosts of M. incognita (Kofoid & White, 1919) Chitwood 1949 and M. chitwoodi Golden et al. (1980) Mojtahedi et al., 1993; Curto et al., 2012 is also attributable to the presence of cyanogenic compounds.

The increased expression of the Mh–hsp90, Mh–hsp1 (Hsp90 and Hsp70 protein family), and Mh–hsp43 (sHsp protein family) genes observed in J2 stage specimens was a component of the body’s response to environmental stress conditions such as common vetch (cv. Ina) seed diffusates. It was also observed that the increase in the level of expression of these genes was accompanied by the motility loss in J2 stage specimens. Our observations are consistent with previous literature reports regarding the chaperone role of the indicated hsp90 and hsp1 genes in the protection of cells against oxidative stress and their involvement in maintaining homeostasis Kegel et al., 1996; Wang et al., 2004; Haslbeck et al., 2005; Ungelenk et al., 2016; Balchin et al., 2016; Fernandez-Fernandez et al., 2017; Schopf et al., 2017; Craig, 2018; Nillegoda et al., 2018; Mok et al., 2018; Janowska et al., 2019; Doyle et al., 2019; Genest et al., 2019. Not only our study, but also other studies concerning three species of Meloidogyne nematodes: M. artiellia, M. incognita and M. hapla, have shown that changes in the environment of these nematodes also caused changes in the expression of hsp90 gene. A significant increase in expression of this gene was observed as a result of thermal (heat and cold) stress, heavy metals stress, and inorganic compounds Bai et al., 2014; De Luca et al., 2009; Wu et al., 2019a. The marked increase in the expression of the hsp90 gene as a result of the action of various environmental stressors implies that this gene can be used as a potential bioindicator of the environmental impact on the organism of Meloidogyne nematodes Bai et al., 2014. The hsp1 gene encodes the Hsp70 protein in the C. elegans, which is critically required during early juvenile stage in the development of this nematode Papsdorf et al., 2019. The research showed that overexpression of hsp1 leads to locomotion defects in C. elegans Papsdorf et al., 2014. Our research also shows an increase in expression of Mh–hsp1 gene and a decrease in motor activity in M. hapla. However, additional studies are needed to confirm this relationship. According to Bai et al. (2014), Hsp90 as a potential bioindicator should be applied simultaneously with Hsp70 to reflect the environmental influence on organisms more accurately.

The highest increase in expression as a result of exposure of the J2 stage of M. hapla to the common vetch seed diffusate of cv. Ina was observed in the Mh–hsp43 gene. The study shows an association between the increased expression of Mh–hsp43 gene and the adverse effect of common vetch seeds (cv. Ina) with a high content of cyanogenic compounds. These genes encode proteins belonging to the small heat shock proteins (sHsp). sHsp is a family of molecular chaperones characterized by a low molecular weight of 12–43 kDa. The primary function of sHsp is to maintain protein homeostasis in response to a variety of stress conditions, including heat shock, hyperosmosis, starvation, and oxidative stress Haslbeck et al., 2005; Janowska et al., 2019; Kegel et al., 1996; Ungelenk et al., 2016; Wang et al., 2004. Hsp43 expression was observed in all developmental stages of C. elegans (Ding and Candido, 2000). In C. elegans, the Hsp43 protein has been shown to be expressed in the epidermis and plays an important role in the resistance of this organism to heat stress (Liu et al., 2018; Fu et al., 2020). The tested expression of the Mh-hsp43 gene was 1.6 times higher than in the control sample, which indicates a significant role of this gene in the response to oxidative stress caused by the exposure of the J2 stage of M. hapla to the diffusate from V. sativa seeds cv. Ina.

Unlike with the Mh–hsp90, Mh–hsp1, and Mh–hsp43 genes, we did not observe an increase in the expression level of the Mh–hsp60 and Mh– hsp12.3 genes in the study. The Mh–hsp60 gene is responsible for encoding Hsp60 protein, which is probably involved in the protection of the mitochondria against the negative effects of heat stress Ewalt et al., 1997; Xu et al., 2010 and is indicated as a potential biomarker of toxic stress in nematodes Kammenga et al., 1998. The increase in Hsp60 protein synthesis in nematode Plectus acuminatus Bastian 1865 treated with copper or cadmium, as well as unaltered expression levels of Mh–hsp60 in J2 of M. hapla treated with common vetch seed diffusates, suggests that Mh–hsp60 gene is not involved in nematode stress response to HCN. We conjecture that the concentration of HCN in the environment was too low or the exposure time was not long enough to induce a marked increase in the expression of Mh–hsp60 gene. It can be assumed that the absence of an increase in the expression level of Mh–hsp12.3 gene may also be down to the same factors as mentioned for Mh–hsp60 gene.

Our study has demonstrated the negative impact exerted by common vetch seeds on the motility of the northern root-knot nematode J2 stage. The behaviour of the J2 stage, characterized by the loss of motility in these nematodes, was accompanied by an increase in the expression of the stress genes Mh–hsp90, Mh–hsp1, and Mh–hsp43. The hsp90 gene could be a potential bioindicator of the effects of the environment on the Meloidogyne organism. The admixture of common vetch seeds to arable and horticultural soils could become an effective method of limiting M. hapla population growth and its damage potential to crops. However, apart from being highly effective, it should be proven to not adversely affect the crop itself and, last but not least, to not pose a risk to food safety. Introducing common vetch seed as a soil additive may contribute to reducing weed growth as well as limiting nematode development in weed roots.