Considering that both A2-J and A2-O induced the expression of suberin biosynthetic genes, the expression of these genes was possibly up-regulated by a generalized wounding signal during nematode infection. We therefore investigated the expression levels of the wound-responsive genes Arabidopsis thaliana activation factor 2 , which encodes a wound-responsive NAC transcription factor , and pathogen-related 4 . Not surprisingly, the expression patterns of wound-responsive genes were similar to those of the suberin biosynthetic genes. These results suggest that infection with A2-O may cause more wounding and damage than A2- J, but that the wounding signal generated during the earliest events in nematode infection, whether a resistant or susceptible reaction occurs later, may induce the expression of suberin biosynthetic genes. Considering the suberin biosynthetic gene transcription data, we predicted that infection with A2-O or A2-J would induce the accumulation of suberin. To test our prediction, we examined and measured the chemical composition of aliphatic suberin at the nematode infection site, but total aliphatic suberin content did not increase after A2-J or A2-O infection . However, the abundance of single monomers, ω-hydroxy acid C16 and α,ω-diacid C16, were significantly higher after infection with A2-O than mock treatment . In this study we established an experimental system using a single cultivar of S. torvum and two pathotypes of RKNs M. arenaria A2-J, flower display buckets which is able to infect and establish a parasitic relationship with the plant, and A2-O, which induces a resistance response.
To the best of our knowledge, this is the first comparative RNA-seq analyses in a single cultivar with virulent or avirulent nematodes. Using this experimental system, we were able to catalog changes in gene expression at the very early stage of infection with a high degree of sensitivity compared to previous studies . Because there are clear morphological differences in root tips infected with A2-J and A2-O within 4 DPI, the sum of host responses by this stage of infection likely has determined the outcome of infection. Interestingly, at 1 DPI, A2-O induced the expression of genes encoding class III peroxidases, fatty acid desaturases, and enzymes involved in isoprenoid biosynthesis , whereas A2-J had not induced any statistically significant changes in the expression of defense response genes . These results suggest that A2-J initially evades recognition by S. torvum and/or actively suppresses the induction of transcriptional changes in the host. Our results also show that A2-J induces the expression of genes associated with susceptible responses that are related to gall formation at around 2–3 DPI . The delay in expression of susceptibility-associated genes is consistent with the length of time it takes for nematodes to migrate through the plant’s vascular system to their target cells. Sampling only infected root tips reduced background plant transcripts and enabled us to detect dynamic changes in gene expression that are directly associated with the establishment of a parasitic relationship or with active host defense. The range of calculated logFC values of root tip genes expressed at 3 DPI with A2-O was from −8 to 12.8 , which is quite broad for very early stages of infection.
For example, in previous transcriptome analyses using S. torvum during infection with an avirulent isolate of M. incognita, chitinase expression in whole roots was only 2–4 times higher upon infection than in uninfected roots . In contrast, our analyses showed that the expression of chitinases in the root tips infected with A2- O at 3 DPI was several hundred times higher than in the mock treatment, clearly demonstrating the high sensitivity of this method for host-parasite transcriptomics . This level of sensitivity allowed us to identify genes that had not previously been connected with resistance or susceptibility in this system.The cell walls of giant cells require thickening and loosening to allow cell expansion that increases the surface area of the plasma membrane, and to support nutrient uptake by the nematodes . The cell walls of giant cells and syncytial feeding sites induced by CNs contain high-ester pectic homogalacturonan, xyloglucan, and pectic arabinan , suggesting that these polysaccharides are responsible for the flexible properties of feeding site cell walls. Our transcriptome results confirm the importance of cell wall modification enzymes during gall formation. In addition to cell wall modification enzymes, infection with A2-J specifically induced the expression of spermidine synthase . Interestingly, a virulence effector protein secreted from CNs, 10A06, functions through its interaction with Arabidopsis spermidine synthase 2 by increasing spermidine concentrations, subsequently increasing polyamine oxidase activities. An increase in polyamine oxidase activity results in the induction of cellular antioxidant machinery in syncytia and disruption of SA-mediated defense signaling. Although there is no clear homolog of 10A06 in RKNs such as M. incognita, it is possible that there is a functional ortholog of 10A06 that up-regulates the expression of spermidine synthase.
Potentially, transcriptional activation of the spermidine synthase gene by PPNs may turn out to be a common strategy for suppressing plant immunity.Infection with A2-O, but not with A2-J, strongly induced genes encoding sesquiterpene synthases . High levels of expression of these genes occurs at the very early stages of infection , which sets these genes apart from the other genes induced by A2-O infection , suggesting the importance of sesquiterpenes as an early line of defense against PPNs. Capsidiol, a sesquiterpene, is the major phytoalexin produced in the Solanaceae plants Nicotiana spp. and Capsicum spp. in response to fungal and bacterial infection . Capsidiol is toxic to many oospore and fungal pathogens, such as Phytophthora capsici and Botrytis cinerea , and suppresses the mobility of false rootknot nematode Nacobbus aberrans . We found that A2-O induces the expression of CYP71D7-like protein, the closest homologue to CYP71D20 from Nicotiana benthamiana, which converts 5-epiaristolochene to capsidiol . A2-O also induces the expression of genes encoding 5-epiaristolochene synthase, which converts farnesyl diphosphate to 5-epiaristolochene . Although capsidiol production is not common in Solanum spp., S. torvum may produce similar sesquiterpene derivatives that are toxic to PPNs. Another sesquiterpene that may be involved in resistance is the phytoalexin solavetivone, because A2-O induces the expression of genes encoding vetispiradiene synthase and premnaspirodiene oxygenase, which sequentially convert farnesyl diphosphate to solavetivone via vetispiradiene . Although the nematicidal activity of solavetivone has not yet been reported,and membrane transport, chalcone synthase, and spermidine synthase at an early phase of gall formation.In Cluster 8 , GO terms that were significantly enriched were related to defense responses, including “defense response to fungus ”, “defense response to bacterium ”, “killing of cells of other organism ”, and “regulation of salicylic acid biosynthetic process ”. In addition, GO terms involved in lignin biosynthesis, including “lignin biosynthetic process ” was also overrepresented in Cluster 8 . In Cluster 9, the significantly enriched GO terms were related to biosynthesis of isoprenoids ”, “terpenoid biosynthetic process ”, and “farnesyl diphosphate catabolic process ” . We also found that the genes that are highly expressed after infection with A2-O in Cluster 8 and 9 include defense-related genes encoding chitinase, β-1,3-glucanase, flower bucket and serine protease inhibitor, sesquiterpene synthase, fatty acid desaturase 2 , ferulic acid 5-hydroxylase which is involved in lignin biosynthesis, berberine bridge enzyme -like protein, which is involved in oxidation of cinnamyl alcohol . Fatty acids are major and essential components of all plant cells and are also precursors for a variety of plant metabolites, including signaling molecules and phytoalexins . FAD2 encodes 1 12-desaturase that catalyzes the conversion of oleic acid to linoleic acid . The Arabidopsis genome has only a single FAD2 gene , but most other plant species carry multiple FAD2 homologs . The duplication of FAD2 genes in plants would have enabled the functional diversification of these enzymes, leading to divergent catalytic activities and the synthesis of novel metabolites. For example, recent studies have shown that tomato has non-canonical FAD2 family proteins that lack 1 12-desaturase activity . In particular, ACET1a/b and FAD2-9 are noncanonical FAD2 involved in the biosynthesis pathway from linoleic acid to a phytoalexin, falcarindiol . Falcarindiol has not only anti-bacterial and anti-fungal activities but also nematicidal activity to M. incognita and pinewood nematode Bursaphelenchus xylophilus .
Infection with A2-O rapidly induced the expression of ACET1a/b and FAD2-9 , suggesting that infection with A2-O rapidly activates a biosynthesis pathway similar to the falcarindiol pathway, but the production of falcarindiol by S. torvum needs to be experimentally confirmed in the future. The GO enrichment analysis of Cluster 8 revealed that “lignin biosynthetic process ” was significantly enriched , and that the expression of F5H in Cluster 8 was very high andspecifically induced after infection with A2-O . Lignin is a phenylpropanoid polymer that is deposited predominantly in the secondary cell wall, making the cell wall rigid and impervious to water . Lignin polymer is synthesized via oxidative combinational coupling of lignin monomers , namely p-coumaryl alcohol, sinapyl alcohol, and coniferyl alcohol. The lignin subunits constituted by these monolignols are p-hydroxyphenyl , syringyl ,and guaiacyl groups, respectively. All of the monolignols are synthesized from phenylalanine through the general phenylpropanoid and monolignol-specific pathways . Normally, lignin deposition occurs in the root endodermis of the differentiation zone and constitutes the Casparian strip, which functions as a physical barrier that prevents free diffusion of solutes and ions between the xylem and the soil . However, biosynthesis and deposition of lignin can be induced in response to biotic stresses , which prompted a closer examination of the expression patterns of genes involved in the lignin biosynthetic pathway whose expression was up-regulated by infection with either A2-J or A2-O . Infection with A2-O induced the expression of genes encoding phenylalanine ammonia-lyase , cinnamate 4-hydroxylase , 4-coumaroyl-CoA ligase , p-hydroxycinnamoylCoA:shikimate p-hydroxycinnamoyl transferase , caffeoyl-CoA O-methyltransferase , cinnamoylCoA reductase , F5H, caffeic acid O-methyltransferase , and cinnamyl alcohol dehydrogenase . Infection with A2-J also induced the expression of some of these genes, but to a much lesser extent than A2-O . Phloroglucinol staining of infected roots allowed us to visualize the intensity and location of lignin accumulation . Infection with A2-O, but not with mock treatment, induced ectopic accumulation of lignin in root tips. With A2-J infection, the area of the root proximal to gall tissue was very slightly stained with phloroglucinol, but the gall itself had little or no detectable phloroglucinol staining. These differences in lignin staining intensity may reflect differences in the expression of lignin biosynthetic genes after infection with A2-J and A2-O .There was no enrichment of specific GO terms in Cluster 7 . However, we found that both A2-J and A2-O strongly activate the expression of suberin biosynthetic genes, including aliphatic suberin feruloyl transferase , cytochrome P450 86A1 , cytochrome P450 86B1 , glycerol- 3-phosphate acyltransferase 5 , and β−ketoacyl−CoA synthase , but A2-O induced slightly higher expression of these genes than A2-J. Suberin is a cell wall component that restricts water loss, nutrient elution, and pathogen infection . It is normally deposited in the cell walls of endodermal cells, but not in the root tip , and several reports showed that suberin synthesis is induced in wounded tissues . Considering that both A2-J and A2-O induced the expression of suberin biosynthetic genes, the expression of these genes was possibly up-regulated by a generalized wounding signal during nematode infection. We therefore investigated the expression levels of the wound-responsive genes Arabidopsis thaliana activation factor 2 , which encodes a wound-responsive NAC transcription factor , and pathogen-related 4 . Not surprisingly, the expression patterns of wound-responsive genes were similar to those of the suberin biosynthetic genes. These results suggest that infection with A2-O may cause more wounding and damage than A2- J, but that the wounding signal generated during the earliest events in nematode infection, whether a resistant or susceptible reaction occurs later, may induce the expression of suberin biosynthetic genes. Considering the suberin biosynthetic gene transcription data, we predicted that infection with A2-O or A2-J would induce the accumulation of suberin. To test our prediction, we examined and measured the chemical composition of aliphatic suberin at the nematode infection site, but total aliphatic suberin content did not increase after A2-J or A2-O infection . However, the abundance of single monomers, ω-hydroxy acid C16 and α,ω-diacid C16, were significantly higher after infection with A2-O than mock treatment .