Host-induced gene silencing of specific genes of Plasmodiophora brassicae as an approach to control clubroot disease

doi:10.21203/rs.3.rs-4877911/v1

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Host-induced gene silencing of specific genes of Plasmodiophora brassicae as an approach to control clubroot disease

https://doi.org/10.21203/rs.3.rs-4877911/v1

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Clubroot disease, caused by the biotrophic pathogen Plasmodiophora brassicae, poses a significant threat to global cruciferous crops production. Current prevention and control strategies are ineffective against P. brassicae. Therefore, new control approach is needed. We have identified two putative effectors, Pb48 and Pb52, which possess secretory functions and exert influence on plant defense. Instantaneous expression of hairpin RNAi constructs with sequence homology to P. brassicae effector Pb48 or Pb52 was performed in Brassica rapa. The successful expression in host and their uptake by P. brassicae were confirmed through observation of green fluorescence in root hair and root epidermal cells as well as within P. brassicae zoosporangia. Successful silencing of either Pb48 or Pb52 resulted in reduced root gall size and enhanced resistance of the host to P. brassicae infection. Especially, silencing of Pb48 led to a decrease in zoosporangia numbers within root hair and epidermal cells. Silencing either Pb48 or Pb52 also resulted in decreased expressions of cytokinin biosynthesis gene IPT1 and auxin homeostasis GH3.5 associated with hormone regulation pathways involved in clubroot development. The transient expression of short interfering RNAs from P. brassicae demonstrates its potential as an effective strategy against this pathogen, thereby paving the way for future developments that confer disease resistance to susceptible host.

host-induced gene silencing

Plasmodiophora brassicae

resistance

effector

Clubroot disease, caused by Plasmodiophora brassicae, is the most devastating disease of cruciferous crops worldwide (Kageyama and Asano, 2009). Clubroot disease has been reported in at least 80 countries, and need to be wary of its expansion to new regions and cause more serious economic losses (Ludwig-Müller et al. 2022). The pathogen can infect various cruciferous crops, such as oil crops (rape seed), vegetables (cabbage, Chinese cabbage, cauliflower, turnip, radish) and fodder crops (swedes and fodder turnip). P. brassicae can infect the cortical cells of susceptible host and further affect the entire root system.

Various management strategies for clubroot disease have been researched and applied. Fungicides are implemented in fields infected with P. brassicae, however, no single fungicide can be used as an effective prevention and control. P. brassicae is a biotrophic obligate parasite. Crop rotations are recommended as a management strategy. Due to P. brassicae resting spores can maintain vitality in the soil for many years in the absence of suitable host (Peng et al. 2009), it means that a field of severe infection may be permanently unsuitable for Brassica crops cultivation. Clubroot disease become a significant disease, especially for farmers in Asian, where cruciferous species occupy a relatively large percentage of vegetables due to the importance in their cuisine. Crop rotation, fungicide application, increased soil pH and improved drainage conditions play a certain role in prevention against clubroot disease. The genetic resistance through traditional crop breeding is an acceptable and popular approach against plant disease. Due to the presence of multiple races within root gall and the inherent variability of P. brassicae, genetic resistance has proven to be somewhat unstable for P. brassicae (Porth et al., 2003). In addition to traditional crop breeding approach, genome editing has been in practice for giving more durable resistance to pathogens that are prone to variation.

At present, the fundamental theoretical research on the pathogenicity of P. brassicae is limited. However, with the release of genome sequence of the German SSIe3 as the “reference genome of P. brassicae” (Schwelm et al. 2015), the pathogenic determinants has been gradually identified, such as effectors, which play a crucial role in regulating host defense responses (Toruno et al. 2016). However, their functions remain largely unknown. Therefore, it is need to develop a method for functionally characterizing putative pathogenicity determinants in P. brassicae. Unveiling their functions will aid in establishing corresponding prevention strategies.

The RNA interference (RNAi) technology can specifically silence a target gene via a double stranded RNA (dsRNA) molecule with its complementary mRNA (Fire et al. 1998). Therefore, we can utilize the host plants to generate siRNAs that match the gene of the invading pathogen to silence the targeted gene (Dou et al. 2020; Wang et al. 2020). An approach called host-induced gene silencing (HIGS) has been successfully used in developing host resistance against pathogens (Zotti et al. 2017). The efficiency of HIGS is based on the ability of pathogen to take double-strand or siRNA from plants (Baulcombe, 2015). The successful assimilation and processing of dsRNA has been observed in various plant pathogenic fungi, such as Botrytis cinerea (Wang et al. 2016), Puccinia striiformis (Qi et al. 2018) and Magnaporthe oryzae (Zhu et al. 2017; Guo et al. 2019), etc. Considering the ability of P. brassicae to uptake nutrients from host cells via phagocytosis, it is plausible that siRNA or protein of transient expression in plants can be uptaken by P. brassicae. HIGS mediated- resistance might be appropriate for P. brassicae.

Considering the efficacy of HIGS in silencing specific genes of pathogen dependents on the cross-kingdom exchange of RNAs between the plant and interactional pathogen, as well as the response of different host-pathosystems, therefore, it is essential to assess the efficacy of HIGS in a particular disease. In this study, we first aimed to establish and demonstrate the effect of HIGS in the P. brassicae-Brassica pathosystem. In view of the fact that almost all cruciferous crops are the hosts of P. brassicae, we intend to establish a more extensive, convenient and suitable approach for combating P. brassicae. Agrobacterium-mediated transient expression is a common method, such as agroinfiltration in leaves of Nicotiana benthamiana. In this study, we establish a method of transient expression in the seedlings, and ascertained the potential of HIGS in Brassica rapa and Brassica napus to target specific genes of P. brassicae. Selection of target gene is the key step for RNA silencing. Pathogen effectors play a crucial role in successful infection (Presti et al. 2015). Similar to other plant pathogens, P. brassicae possesses the characteristics of secreted proteins (Perez-Lopez et al. 2020), and the secretory activity of some putative secreted proteins has been determined. Here, two effectors from P. brassicae were selected as target genes based on our previous identification (Yang et al., 2022).

In view of the limited control measures of clubroot disease, it requires multifaceted approaches to management. New alternatives need to be evaluated. In this study, we tried to establish a way of HIGS through agrobacterium-mediated transient expression in controlling clubroot disease. On the one hand, it will allow us to conduct further research on its pathogenesis, on the other hand, it will perform as a convenient, non-transformative, environment-friendly and potential pathogen management approach to protect crop against P. brassicae.

Preparation and inoculation of resting spores P. brassicae was obtained from Chengdu in Sichuan Province of China (Zheng et al. 2019), and was identified as pathotype 4 using the differential system of Williams (1996). P. brassicae resting spores were prepared as described (Ji et al. 2014). The concentration of resting spores was adjusted to 1×10⁷ spores/ml for inoculation, 300 µL suspension was dripped into the soil around the root of each plant.

Morphological characterization of P. brassicae The seedlings inoculated with P. brassicae for about 7–14 days were pulled out to wash off the soil on the roots. The roots were stained with 0.5% fluorescent pink dye for 1–2 min, the background color of roots was removed with sterile water. The plasmodia, zoosporangia and resting spore of P. brassicae can be stained red. The infection in root hair and epidermal cell was directly observed under Carl Zeiss microscope (Germany). To visualize resting spores within root galls, thin slices were prepared using the hand-slicing method and examined under the microscope.

Assessment of secretory activities directed by signal peptides from putative secretory proteins in yeast High-fidelity DNA polymerase (TransGen Biotech, Beijing, China) was used to amplify the signal peptide of P. brassicae effector Pb48 and Pb52. The primer sequence are showed in Supplementary Table 1. Purified DNA was separately ligated into the pSUC2 vector, and then transformed into the yeast strain YTK12. Secretory activity was assessed using growth assays (Jacobs et al., 1997). The transformed YTK12 strains with secretory activity could grow on CMD-W medium (0.67% yeast N base without amino acids, 0.075% tryptophan dropout supplement, 2% sucrose, 0.1% glucose, and 2% agar) and YPRAA medium (1% yeast extract, 2% peptone, 2% raffinose, and 2 µg/mL antimycin A). The invertase activity was determined by the color variation of colourless 2,3,5-triphenyltetrazolium chloride (TTC) to insoluble, red-colored 1,3,5-triphenylformazan (TPF).

Pb48 and Pb52 transient expression in N. benthamiana The full-length nucleotide sequence was amplified by TransStart FastPfu Fly DNA Polymerase (TransGen Biotech, Beijing, China). The primers design for Pb48 and Pb52 were based on P. brassicae PBRA_005225 (Accession: CDSF01000068.1) and PBRA_004448 (Accession: CDSF01000035.1), respectively. PCR products were inserted into the pBIB-BASTA-35S-GWR-green fluorescent protein (GFP) vector. The primer sequences are listed in Supplementary Table S1. The recombinant vector were transferred into A. tumefaciens GV3101. Then A.tumefaciens GV3101 were cultured and centrifuged, the precipitation was resuspended in MMA infiltration solution (10 mM MgCl₂, 10 mM MES and 150 mM acetosyringone). Nicotiana benthamiana leaves were infiltrated at the four-leaf stage.

The construction and transient expression of HIGS vector The 369-bp partial Pb48 forward fragment and the 305-bp partial Pb52 forward fragment were individually inserted into the XhoI/KpnI site of the pKANNIBAL vector, and then the corresponding reverse fragment was individually inserted into the HindⅢ/XbaⅠ site of the same vector. The upstream sequences of the forward fragments and downstream of the reverse fragments had the BsrGI restriction enzyme site. The fragments released by BsrGI digestion were inserted into the BsrGI/BsrGI site of pBIB- BASTA-35S-GWR-GFP vector, namely Pb48-HIGS and Pb52-HIGS. The recombinant vectors were transferred into A. tumefaciens strain GV3101. The transformed A. tumefaciens cells were cultivated and centrifuged. The precipitation was resuspended in the MMA solution (10 mM MgCl₂, 10 mM MES and 150 mM Acetosyringone, PH5.7).

The susceptible Brassica napus cultivar Chuanyou 36 and Brassica rapa cultivar Zaoshu 5 were used in this experiment. Fifty seeds were mixed in the above preparative solution and gently shake for 10–20 h (Zhang et al. 2023). The treatment seeds were sown in sterilized soil, and grown in a 25℃ incubator with a 16h light/8 h dark. 500 µl above solution was dripped onto the soil surrounding the roots of six-days-old seedling. Roots of 19 days-old seedlings were collected to determine silencing efficiency levels.

Seven-days-old seedlings were inoculated with P. brassicae. A 300 µl suspension containing resting spores was dripped into the soil around the root of each seedling. The disease index was determined at 25–30 dpi. For each experiment, 50 plants were assessed for clubroot severity (disease severity index, DSI), according to a standard 0–4 scale (Siemens et al. 2002).

Fluorescence observation in the roots of B. rapa and P. brassicae The seeds were treated with transformed A.tumefaciens GV3101 carrying 35S-GFP, 35S-Pb48- HIGS-GFP and 35S-Pb52-HIGS-GFP, respectively. The seeds were planted in petri dishes. Four-days seedlings were used to GFP fluorescence analysis and inoculated with P. brassicae. Six-days seedlings were treated with transformed A. tumefaciens GV3101 again. At 7 days post-inoculation with P. brassicae, GFP fluorescence was observed. The vector-encoded GFP possessed its own start codon enabling independent expression or co-expression with its syncretic gene.

Quantitative real-time reverse transcription-polymerase chain reaction Total RNA was extracted from B. rapa roots as previously described (Yang et al. 2022). qRT-PCR was conducted on a Bio-Rad CFX96 Real-Time PCR System (Bio-Rad, USA). The actin in B. rapa was used as an internal reference gene to determine the values for the relative expression levels. We used the 2^−∆∆Ct algorithm to calculate the relative expression levels of target genes (Livak et al. 2001). Three replicates were used. The primer sequences are listed in Supplementary Table S1. Statistical analysis was performed by one-way ANOVA and followed by Tukey’s multiple comparison test.

Pb48 and Pb52 effect plant immunity response To successfully colonize hosts, pathogen secrete effectors into plant to operate different virulence strategies. Two putative effectors Pb48 and Pb52 lacking a signal peptide (SP), were individually cloned into the plant expression vector. We analyzed the effect on the plant defense response. In the agroinfiltration bioassays, Pb52 could trigger cell death while Pb48 suppressed BAX-induced cell death in Nicotiana benthamiana leaves (Fig. 1). BAX is a mammalian pro-apoptotic gene which is known to trigger cell death in plants (Dong et al., 2008).

Pb48 and Pb52 is secreted via the classical secretory pathway To validate the secretory function of signal peptide (SP), a SP trap system was employed in yeast. Yeast strains expressing SUC2 fused with the signal peptide could enable YTK12 growth on both CMD-W and YPRAA medium. Moreover, transformed yeast strains carrying either Pb48 or Pb52 exhibited secretion of invertase, which was capable of converting colorless TTC into insoluble red TPF (Fig. 2). The result indicates that Pb48 and Pb52 are secreted via the classical pathway.

The RNAi vectors of Pb48 and Pb52 work well in B. rapa and P. brassicae To design the RNAi constructs that could silence Pb48 and Pb52, a forward and reverse DNA fragment of Pb48 or Pb52 was individually inserted into the polyclonal sites of RNAi vector pKANNIBAL to generate a dsRNA sequence with a hairpin structure. Then the fragments were digested and ligated into the 35S-Basta-GFP vector (Namely Pb48-HIGS and Pb52-HIGS) (Fig. 3). Agrobacterium tumefaciens GV3101 carrying the recombinant vector was used to treat B.rapa seeds, followed by sowing them in sterilized soil.

P. brassicae is an obligate parasite, and it is difficult to perform for over- expression or gene knockout in P. brassicae itself. P. brassicae obtain nutrients through phagocytosis, therefore, the products of siRNA or protein by transient expression in plants might be uptaken by P. brassicae. In view of the fact that transient expression is performed by infiltrating the seeds and roots, which is different from the infiltration in the leaves, we verified whether the gene could transient expression in the roots and whether P. brassicae could swallow these proteins by observing fluorescence under microscope. Fluorescence was obviously observed in the roots at 3 days after planting (Fig. 4). At 7 dpi, P. brassicae developed into the typical zoosporangium, and fluorescence was clearly observed in the zoosporangium (Fig. 4), indicating that P. brassicae might phagocytose these proteins.

Next, we quantified the transcription level of two genes by qRT-PCR at 12 days post-inoculation with P. brassicae. The expression of Pb52 was significantly suppressed, Pb48 was down-regulation (Fig. 5). The results demonstrated that RNAi constructs of two target genes were successfully processed in transient expression plants, thereby reducing the transcript levels of two genes in the invading of P. brassicae.

The host-induced gene silencing of Pb48 or Pb52 enhance plant resistance to P. brassicae At 7 dpi and 14 dpi, the roots of cabbage were took and stained with fluorescent peach dye to observe P. brassicae under microscope. The numbers of zoosporangia in the root hair and root epidermal cells were less in the seedlings of silencing Pb48 than the other seedlings at 7 dpi (Fig. 6). Pb52 was silenced, there was no difference in the number of zoosporangia when compared with the control-treated seedlings. At 14 dpi, empty zoosporangia gradually appeared, indicating the release of secondary zoospores. The empty zoosporangia in the seedlings of Pb48 and Pb52 silencing were less as compared with the seedlings of control treatment (Fig. 6), indicating that Pb48 and Pb52 affected the development of P.brassicae.

At 36 dpi, the diseased roots were sliced for microscopic observation. The resting spores in the root cortical cells of Pb48 and Pb52 silencing seedlings was relatively dispersive than that in the control treatment seedlings (Fig. 7). At 36 dpi, the plant incidence and disease index of Chinese cabbage were counted. About 50 plants were taken from each treatment for statistics. It was found that the symptoms of Chinese cabbage after silencing treatment were significantly mild than those of the control group (Fig. 7). The incidence and the disease index of root disease were 93.33% and 66.67, 71.74% and 33.85, 62.75% and 30.81 for control treatment group, Pb48 silencing group and Pb52 silencing group, respectively (Table 1). The data uncover a role of Pb48 and Pb52 in P. brassicae virulence. HIGS in Brassicae napus, can also mediate resistance to P. brassicae.

Taken together, our results suggest that HIGS targeting the specific virulence genes in P. brassicae can be used as a approach for developing P. brassicae management.

The silencing of Pb48 or Pb52 down-regulate the gene expression of auxin and cytokinin pathway In view of the fact that the symptom formation in seedlings of Pb48 and Pb52 silencing was different from the control treatment, which might be related to the effect of host defense or growth hormone pathway. The detection results showed that the expression levels of defense pathway marker genes PR1 and PDF1.2 were not significantly different (data did not shown), however, the expression levels of auxin pathway gene GH3.5 and cytokinin pathway gene IPT1 were significantly decreased (Fig. 8). Both auxin and cytokinin have key regulatory role in the infected root tissues of P. brassicae (Prerostova et al. 2018).

P. brassicae is a typical soil-borne pathogen that causes massive yield losses of cruciferous plants all over the world. Due to the widespread planting of cruciferous crops and rapid loss of resistance to clubroot disease, as well as the presence of multiple races of P. brassicae in the root gall, the application of disease-resistant varieties is limited. In the soil, resting spores easily accumulate to a level which cause substantial impact on crop development. The crop rotation, chemical control and agronomic practices have not been considered as the efficient tools for clubroot managements. Therefore, new control approach is needed.

The host-induced gene silencing (HIGS) may be a powerful and environmentally-friendly approach to control crop disease by silencing the essential pathogenic genes of invading pathogen. HIGS is a development of virus induced gene silencing (VIGS). The virus-mediated HIGS in planta-generated RNAi, such as tobacco rattle virus (TRV), bean pod mottle virus (BPMV) and barley stripe mosaic virus (BSMV), has been developed to serve as a strategy for controlling plant disease (Song and Thomma, 2016; Panwar et al. 2017; Qi et al. 2018). For example, TRV-based HIGS silenced Ave1 in Verticillium dahliae and enhanced the host resistance to Verticillium wilt disease (Song and Thomma, 2016). RNAi-based HIGS technology provides an innovative approach to control wheat and barley diseases, such as Blumeria graminis, Puccinia striiformis, Fusarium species, etc (Qi et al. 2018). Otherwise, the expression of RNAi constructs including a dsRNA sequence of hairpin structure in plant also could generate small interference RNA to target the pathogen gene and enhance resistance against phytopathogen (Panwar et al. 2018; Chen et al. 2022). HIGS assays have been successfully applied. However, the tool is not applied to control P. brassicae.

After a GFP-reporter system Pb48-HIGS-GFP and Pb52-HIGS-GFP treatment, the fluorescence signal in the roots was observed, which indicated that the treatment method of transient expression is feasible. The fluorescence signal in the intra-cell of P. brassicae was also observed, which suggested that proteins of Pb48 and Pb52 might be uptaken by P. brassicae. In the meantime, the recombinant vectors of Pb48 and Pb52 or siRNAs might be uptaken by P. brassicae. SiRNA, microRNA (miRNAs) and long dsRNA produced within the host plant has been shown to be able to be uptaken by filamentous fungi (Weiberg et al. 2013; Wang et al. 2016). In this study, the fluorescence signals verified the cross-kingdom transfer between P. brassicae and plants, which suggested that a protozoa, P. brassicae, as it devoured nutrients, was also capable of devouring heterologous proteins and other molecules from plants. The efficiency of HIGS is based on the ability of pathogen to take double-stranded or siRNA from host plants when drawing nutrition (Baulcombe, 2015). The transcripts levels of Pb48 and Pb52 were reduced after HIGS treatment, which revealed that transient expression in hosts could successfully induce the silencing of target gene in P. brassicae.

At present, the transgene-mediated HIGS is the major method of using RNAi (Baulcombe, 2015). Some reports indicated that exogenously applying siRNAs that target pathogenicity genes of pathogen could enhance host resistance (Sarkar and Roy-Barman, 2021; Cheng et al. 2022). In addition, RNAi processes are usually used to resist pathogens of plant leaves. In this study, we established a transient HIGS tool to resist root disease by silencing target genes of invading P. brassicae through pretreating seeds and irrigating roots. Clubroot disease is a seedling susceptible disease, the seedling treatment has an important role in preventing and controlling its harm. The tool represents a promising approach for limiting crop losses caused by P. brassicae, and also offers the functional assay for target virulence genes of P. brassicae. This treatment process also provides reference for other soil-borne disease control.

The selection of effective target genes is key for RNA silencing. The secretory protein plays an important role between plant and pathogen interaction. In addition to establishing the HIGS systems, we identified the secretory activity of Pb48 and Pb52, and ensured the influence on plant immunity. To further understand the effect after silencing Pb48 and Pb52, we determined the expression levels of genes in the auxin and cytokinin pathway. IPT1 and GH3.5 were significantly reduced after Pb48 or Pb52 silencing. IPT1 is a cytokinin biosynthesis gene, which was induced in infected root tissues of P. brassicae (Laila et al. 2020). GH3.5 proteins are involved in the regulation of auxin homeostasis, biotic and abiotic stress, which shows high expression levels after P. brassicae infection (Ludwig-müller et al. 2014). The altered hormonal balance of auxin and cytokinin is the cause of root swelling (Dekhuijzen and Overeem, 1971; Butcher et al. 1974), therefore, we suggested that the development of smaller root gall after silencing of Pb48 or Pb52 was associated with affecting the growth hormone pathways.

In summary, our study showed that the host-induced silencing of effectors by transient expression enhanced host resistance against P. brassicae, which will be a potential for controlling clubroot disease of various cruciferae crops. The approach can contribute to environmentally sustainable agriculture.

Compliance with ethical standards

I certify that this manuscript is original and has not been published and will not be submitted elsewhere for publication. And the study is not split up into several parts to increase the quantity of submissions and submitted to various journals or to one journal over time. No data have been fabricated or manipulated (including images) to support our conclusions. No data, text, or theories by others are presented as if they were our own. The submission has been received explicitly from all co-authors. And authors whose names appear on the submission have contributed sufficiently to the scientific work and therefore share collective responsibility and accountability for the results.

Conflict of interest

The authors declare that they have no conflict of interest.

Human and animal rights

This article does not contain any studies with human participants or animals performed by any of the authors.

Informed consent

Informed consent was obtained from all individual participants included in the study.

Acknowledgments

This study was supported by the State Key Laboratory of Crop Gene Resources Exploitation and Utilization in Southwest China “Biological breeding” Project (SKL-ZY 202222). Natural Science Foundation of Sichuan Science and Technology Agency (2024NSFSC0410). Sichuan Innovation Team Project of National

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Supplementarytable1.xls
Additional file 1: The Supplementary table 1. The primers sequence.

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Host-induced gene silencing of specific genes of Plasmodiophora brassicae as an approach to control clubroot disease

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Declarations

Compliance with ethical standards

Human and animal rights

Informed consent

Acknowledgments

References

Supplementary Files

Status:

Version 1