Periodic Reporting for period 1 - NemHostRangeParadox (A nematode host-range paradox: how can one of the apparently most specialised obligate biotrophic plant-parasites also have the largest host rangeNemHostRangeParadoxA nematode h)
Período documentado: 2021-10-01 hasta 2023-09-30
The root-knot nematode (Meloidogyne incognita) is one of the most important pests worldwide, especially in tropical and subtropical areas of the globe. This single nematode species is able to parasite more than 3,000 different plant hosts including fruits, vegetables and cereals. It exploits the plant resources by using very specialized mechanisms of parasitism. It reprograms the plant cell using small secreted molecules (termed ‘effectors’), culminating in the development of a new pseudo-organ (termed ‘giant cells’), which presents the sole purpose of nourishing the nematode. Although remarkable, it is currently not understood the mechanism underlying this paradox – a specialised parasite presenting the broadest host range of all biotrophic pathogens known.
The HostRangeParadox project aimed to build a holistic view of M. incognita parasitism by analysing the transcriptome profile of the root-knot nematode on ten different host plants. We aimed to identify root-knot nematode’s genes (and consequently effectors) expressed across several hosts or on a specific host. Also, we addressed the host side of the interaction, aiming to identify possible targets (such as metabolic pathways or plant genes) of M. incognita. Finally, our last objective was to confirm our hypothesis: the necessity of some effectors to hosts or in particular interactions and confirm the importance of certain targets in the host x M. incognita interaction.
The HostRangeParadox project aimed to build a holistic view of M. incognita parasitism by analysing the transcriptome profile of the root-knot nematode on ten different host plants. We aimed to identify root-knot nematode’s genes (and consequently effectors) expressed across several hosts or on a specific host. Also, we addressed the host side of the interaction, aiming to identify possible targets (such as metabolic pathways or plant genes) of M. incognita. Finally, our last objective was to confirm our hypothesis: the necessity of some effectors to hosts or in particular interactions and confirm the importance of certain targets in the host x M. incognita interaction.
Our initial hypothesis was that nematode gene expression would be similar in closer related species and, with increasing phylogenetic distance between hosts, it would differ more. The same reasoning was applied to host gene expression. The affected processes by M. incognita would be similar in phylogenetically closely related hosts and differ among distantly related hosts. Therefore, the hosts were selected following some criteria: 1) plants should be susceptible to M. incognita; 2) draft genome, annotation and other resources should be available to them; 3) host’s groups should be spread across the phylogenetic tree of flowering plants to better represent the diversity of hosts; 4) It was also interesting to select more than one host in some groups to check if there was consistency in the transcriptome profile within the group. Following these, a total of 20 hosts were selected and further narrowed down to 10. Then, we started the infection assays (WP-1 and WP-2). The total RNA of uninfected fragments and infected tissues (termed ‘galls’) of host plants were extracted and further analysed. Pre-infective M. incognita J2 was also included in our analysis. Altogether, the data for the 10 hosts and pre-infective J2 totalized 128 raw-read files and 3 Tb of processed and raw data.
The samples were collected at ~ 25 days after inoculation, and at this stage, the females (immature, i.e. J4 and mature) should be the majority of nematodes within the root system. This was later confirmed by our penetration experiments assays. Moreover, at this stage feeding sites are already established and, therefore, the effectors involved are probably related to giant cell development/maintenance, including host defence suppression to keep them alive. Plant resistance to sedentary endoparasitic nematodes often tackles the nematode’s feeding site rather than at the invasion step. If the host defence kills the giant cells, the parasite cannot survive or reproduce.
We found that gene expression of Meloigyne incognita does not follow a clear pattern. The principal components analysis (PCA) grouped the hosts into three groups of expression (Figure 2), in which the group are 1) rice, tobacco, tomato and watermelon; 2) arabidopsis and alfafa; 3) lettuce, maize, melon and safflower. Although related species were in the same group in some cases (tobacco and tomato – both Solanaceae and group 1), this did not happen to others (maize and rice – both Poaceae and group 3 and 1, respectively). When genes were clustered together, two patterns were observed: clusters expressed across different hosts (more numerous) and a few clusters of genes expressed (or dramatically more expressed) in a single host. We obtained the effector candidates by predicting the secretome of M. incognita, and excluding those genes that encode proteins without signal peptide or with transmembrane domain (using SignalP and TMBed, respectively). Our results point out that Meloidogyne incognita tunes gene expression depending on the host. Gene expression defies logic - it does not follow either the phylogeny of the host, physiology of infection, or global gene expression grouping.
Regarding the host-side of interaction, our data shows that the gall is a phenomenon dominated by the repression of genes, i.e. more down-regulated DEGs than up-regulated. Only two exceptions were found. The first was safflower, in which the same number for down and up-regulated was found. However, this could have been influenced by the current status of the genome and annotation available, currently in version 1.0 (Wu et al., 2021). The second case was watermelon, which was found more up-regulated DEGs than down-regulated. The Gene Ontology analysis (GO) revealed that the majority of the processes affected by M. incognita were exclusive to the groups and only a few were shared by them. Regarding down-regulated biological processes, only five (2.3%) are common to all groups: defence response, regulation of the cellular process, regulation of biological process, transmembrane transport, and biological regulation. Regarding up-regulated processes, only two are common to all groups: the mitotic cell cycle process, and the mitotic cell cycle. Both are related to the establishment/maintenance of feeding cells.
In addition to the proposed work, I was also a co-author of a book chapter in a distinguished book in plant pathology (The Agrio's Plant Pathology) and the lead author of a review that addressed several aspects of an emergent plant-parasitic nematode in South America (DOI: 10.1111/ppa.13829). Both materials could be interesting assets to be used in plant nematology.
The samples were collected at ~ 25 days after inoculation, and at this stage, the females (immature, i.e. J4 and mature) should be the majority of nematodes within the root system. This was later confirmed by our penetration experiments assays. Moreover, at this stage feeding sites are already established and, therefore, the effectors involved are probably related to giant cell development/maintenance, including host defence suppression to keep them alive. Plant resistance to sedentary endoparasitic nematodes often tackles the nematode’s feeding site rather than at the invasion step. If the host defence kills the giant cells, the parasite cannot survive or reproduce.
We found that gene expression of Meloigyne incognita does not follow a clear pattern. The principal components analysis (PCA) grouped the hosts into three groups of expression (Figure 2), in which the group are 1) rice, tobacco, tomato and watermelon; 2) arabidopsis and alfafa; 3) lettuce, maize, melon and safflower. Although related species were in the same group in some cases (tobacco and tomato – both Solanaceae and group 1), this did not happen to others (maize and rice – both Poaceae and group 3 and 1, respectively). When genes were clustered together, two patterns were observed: clusters expressed across different hosts (more numerous) and a few clusters of genes expressed (or dramatically more expressed) in a single host. We obtained the effector candidates by predicting the secretome of M. incognita, and excluding those genes that encode proteins without signal peptide or with transmembrane domain (using SignalP and TMBed, respectively). Our results point out that Meloidogyne incognita tunes gene expression depending on the host. Gene expression defies logic - it does not follow either the phylogeny of the host, physiology of infection, or global gene expression grouping.
Regarding the host-side of interaction, our data shows that the gall is a phenomenon dominated by the repression of genes, i.e. more down-regulated DEGs than up-regulated. Only two exceptions were found. The first was safflower, in which the same number for down and up-regulated was found. However, this could have been influenced by the current status of the genome and annotation available, currently in version 1.0 (Wu et al., 2021). The second case was watermelon, which was found more up-regulated DEGs than down-regulated. The Gene Ontology analysis (GO) revealed that the majority of the processes affected by M. incognita were exclusive to the groups and only a few were shared by them. Regarding down-regulated biological processes, only five (2.3%) are common to all groups: defence response, regulation of the cellular process, regulation of biological process, transmembrane transport, and biological regulation. Regarding up-regulated processes, only two are common to all groups: the mitotic cell cycle process, and the mitotic cell cycle. Both are related to the establishment/maintenance of feeding cells.
In addition to the proposed work, I was also a co-author of a book chapter in a distinguished book in plant pathology (The Agrio's Plant Pathology) and the lead author of a review that addressed several aspects of an emergent plant-parasitic nematode in South America (DOI: 10.1111/ppa.13829). Both materials could be interesting assets to be used in plant nematology.
To our knowledge, this is the first attempt to understand it holistically. The data generated by the current project pushes forward our current understanding of root-knot nematode’s parasitism, and it will be a unique reference dataset of M. incognita on different hosts. Also, it will allow the discovery of key components involved in parasitism (e.g. transcription factors and effectors) to be tackled by genome editing tools. I am confident that the data generated on the current project will impact and contribute to the state-of-the-art in the near future. Furthermore, during the MSCA action I was able to promote my research using different media, such as YouTube. I could reach very different audiences - from high school teenagers in a less fortunate municipality of Brazil to researchers from the University of Cambridge.