Periodic Reporting for period 1 - NEMABOX (Deciphering the cyst nematode parasitic program by uncovering how their virulence is orchestrated)
Période du rapport: 2020-04-01 au 2022-03-31
The microbial world contains among the most fascinating biotrophic interactions but also among the most destructive plant pathogens. The study of these complex interactions provides both fundamental knowledge on the molecular dialogue across species from different Kingdoms of Life, but also innovative solutions to fight crop pathogens. The plant parasitic nematodes represent a significant group of plant pathogens that threaten current and future food security. Among them, the cyst nematodes target some of the most important crop species (tomato, potato) and cause up to 80% losses. European regulations ((EC) No1107/2009) increase the stringency of conditions for approval of new chemicals for agriculture, limit the choice of fungicides and pesticides, and prohibit the future use of many effective nematicides. We must improve our understanding of the virulence of the cyst nematodes in order to identify new targets for a sustainable biotechnological control of these pests.
It is known that the parasite breaks the plant cell wall using a stylet to secrete plant cell wall degrading enzymes and inject into the plant cytosol and apoplasm secreted “effector” proteins. Prior to secretion, these effectors are primarily produced in three gland cells in the nematode (one dorsal gland and two subventral) as illustrated overleaf. This “cocktail” of secreted effector proteins allows the cyst nematode to penetrate the plant and manipulate host immunity and physiology.
One of the major questions in the field is, how is this virulence orchestrated at the molecular level? This project significantly improved our understanding of the virulence of cyst nematodes by combining in silico and molecular approaches to decipher their parasitic program.
This action provided two major resources with the lifestage-specific trans-kingdom RNAseq timecourse and the gland cells-specific RNAseq dataset. In addition, it identified a first-of-its-kind subventral gland regulator that regulates the expression of a large set of subventral gland effectors and participate to the plant penetration process. Functional analysis of the SUGR identified two different possibilities of control strategies that can be further investigated to develop impactful solution to cyst nematode infection.
It is known that the parasite breaks the plant cell wall using a stylet to secrete plant cell wall degrading enzymes and inject into the plant cytosol and apoplasm secreted “effector” proteins. Prior to secretion, these effectors are primarily produced in three gland cells in the nematode (one dorsal gland and two subventral) as illustrated overleaf. This “cocktail” of secreted effector proteins allows the cyst nematode to penetrate the plant and manipulate host immunity and physiology.
One of the major questions in the field is, how is this virulence orchestrated at the molecular level? This project significantly improved our understanding of the virulence of cyst nematodes by combining in silico and molecular approaches to decipher their parasitic program.
This action provided two major resources with the lifestage-specific trans-kingdom RNAseq timecourse and the gland cells-specific RNAseq dataset. In addition, it identified a first-of-its-kind subventral gland regulator that regulates the expression of a large set of subventral gland effectors and participate to the plant penetration process. Functional analysis of the SUGR identified two different possibilities of control strategies that can be further investigated to develop impactful solution to cyst nematode infection.
The project started a week after the beginning of the first COVID-19 lockdown in the United Kingdom, in April 2020. The focus was therefore given to the in silico approaches that were not requiring access to the lab. The first work performed was to analyse the lifestage-specific trans-kingdom RNAseq timecourse generated. This timecourse not only provided a reference dataset to the community but also was used to identify co-expression clusters enriched in effector-encoding genes. Meanwhile, several hundred Heterodera schachtii’s gland cells were isolated by collaborators from the Iowa State University and an RNAseq library was generated from the gland cells RNAs extracted. We conducted the gland cells RNAseq analysis and used the gland cell-specific expression dataset obtained to refine our list of candidate regulators, focusing on those presenting a high level of expression in the gland cells.
Once back in the laboratory, an RNAi-mediated silencing of the four most interesting candidates were done before performing a comparative RNAseq analysis in order to identify regulated genes. This allowed the identification of a SUbventral Gland Regulator, coined the SUGR, that was further analysed. The SUGR regulates a set of 297 genes, among them a large set (45%) of gene encoding for secreted proteins, including known and candidate subventral gland effectors. Further analysis were conducted to confirm this hypothesis, among them an in situ hybridization on regulated known and candidate subventral gland effectors in order to localize their expression. The majority of the selected genes were found specifically expressed in the subventral glands, or in both the subventral and dorsal glands. To go further in the analysis of the SUGR and assess its impact on Heterodera schachtii’s pathogenicity, a root penetration assay was performed where SUGR-silenced nematodes were used to infect mustard roots and compared to the appropriate control. This showed the silencing of the SUGR impairs the ability of the nematode to penetrate the plant root and further confirms the SUGR is critically important at the early stage of infection, when the J2s secrete subventral effectors to digest the plant cell wall and enter the plant root.
As it is known that root-derived molecules attract J2s, a similar signal might also activate the SUGR-encoding gene expression, consistent with its early expression during the plant penetration stage. H. schachtii’s juveniles were soaked in mustard roots filtrate before being flash-frozen in liquid nitrogen. SUGR-encoding gene expression was measured by real time qPCR in the root-filtrate and water-soaked nematodes used as a control. This showed the SUGR-encoding gene was activated by the mustard root filtrates, suggesting one or several molecules were detected by the nematode as an pathogenicity activation signal.
Additional analysis performed on the root filtrates showed the activation signal is heat resistant, resistant to proteolysis and has a maximum molecular weight of 3kDa. Biochemical characterization of this root extract may allow the identification of the molecule that activates H. schachtii pathogenicity. Interestingly, soaking the juveniles into root extracts from non-host plants has opposite effect on the SUGR-encoding gene, triggering a repression of its expression, implying H. schachtii can distinguish between host and non-host and responds accordingly.
The identification of a subventral gland regulator and its characterization offers two different possibilities for control strategies. As the genes directly regulated by the SUGR are involved into the plant penetration process, preventing the SUGR from binding to its target genes would directly impact the nematode’s pathogenicity. Alternatively, identifying plant-derived molecules able to repress the SUGR-encoding gene expression would similarly prevent the nematode to efficiently penetrate the root. Being plant-derived, their usage would not be impacted by the current and future EU regulations on the usage of synthetic pesticides as pest control strategies.
Once back in the laboratory, an RNAi-mediated silencing of the four most interesting candidates were done before performing a comparative RNAseq analysis in order to identify regulated genes. This allowed the identification of a SUbventral Gland Regulator, coined the SUGR, that was further analysed. The SUGR regulates a set of 297 genes, among them a large set (45%) of gene encoding for secreted proteins, including known and candidate subventral gland effectors. Further analysis were conducted to confirm this hypothesis, among them an in situ hybridization on regulated known and candidate subventral gland effectors in order to localize their expression. The majority of the selected genes were found specifically expressed in the subventral glands, or in both the subventral and dorsal glands. To go further in the analysis of the SUGR and assess its impact on Heterodera schachtii’s pathogenicity, a root penetration assay was performed where SUGR-silenced nematodes were used to infect mustard roots and compared to the appropriate control. This showed the silencing of the SUGR impairs the ability of the nematode to penetrate the plant root and further confirms the SUGR is critically important at the early stage of infection, when the J2s secrete subventral effectors to digest the plant cell wall and enter the plant root.
As it is known that root-derived molecules attract J2s, a similar signal might also activate the SUGR-encoding gene expression, consistent with its early expression during the plant penetration stage. H. schachtii’s juveniles were soaked in mustard roots filtrate before being flash-frozen in liquid nitrogen. SUGR-encoding gene expression was measured by real time qPCR in the root-filtrate and water-soaked nematodes used as a control. This showed the SUGR-encoding gene was activated by the mustard root filtrates, suggesting one or several molecules were detected by the nematode as an pathogenicity activation signal.
Additional analysis performed on the root filtrates showed the activation signal is heat resistant, resistant to proteolysis and has a maximum molecular weight of 3kDa. Biochemical characterization of this root extract may allow the identification of the molecule that activates H. schachtii pathogenicity. Interestingly, soaking the juveniles into root extracts from non-host plants has opposite effect on the SUGR-encoding gene, triggering a repression of its expression, implying H. schachtii can distinguish between host and non-host and responds accordingly.
The identification of a subventral gland regulator and its characterization offers two different possibilities for control strategies. As the genes directly regulated by the SUGR are involved into the plant penetration process, preventing the SUGR from binding to its target genes would directly impact the nematode’s pathogenicity. Alternatively, identifying plant-derived molecules able to repress the SUGR-encoding gene expression would similarly prevent the nematode to efficiently penetrate the root. Being plant-derived, their usage would not be impacted by the current and future EU regulations on the usage of synthetic pesticides as pest control strategies.
The acquisition of a lifestage-specific trans-kingdom RNAseq timecourse represents a significant advance in our understanding of the cyst nematode pathogenicity, as it allows the study of both the parasite and the host transcriptome in parallel. Combined with a gland cells-specific RNAseq dataset, highlighting the genes highly expressed in this critical organ, those resources now drive forward the state of the art, fostering the identification of key components of the cyst nematodes pathogenicity.
The identification of a subventral gland regulator regulating dozens of subventral gland effectors and participating to the plant penetration process represent a breakthrough in our understanding of the cyst nematode pathogenicity as well as a potential target for future control strategies. Functional analysis of the SUGR also showed it is able to respond to root-derived molecules. The knowledges and resources produced by the NEMABOX action may now be exploited to develop impactful solutions to cyst nematode infections compatible with the EU Regulation on Plant Protection Products.
The identification of a subventral gland regulator regulating dozens of subventral gland effectors and participating to the plant penetration process represent a breakthrough in our understanding of the cyst nematode pathogenicity as well as a potential target for future control strategies. Functional analysis of the SUGR also showed it is able to respond to root-derived molecules. The knowledges and resources produced by the NEMABOX action may now be exploited to develop impactful solutions to cyst nematode infections compatible with the EU Regulation on Plant Protection Products.