These results suggest that S. torvum rapidly induces defense responses against A2-O, which inhibits the maturation of A2- O and gall formation. In contrast, A2-J inhibits or evades the induction of defense responses, continues development, and induces gall formation.RNA-seq analysis was performed to understand the differences in transcriptional regulation of the S. torvum response to infection by nematodes that induce an immune response or that are successful parasites. Eleven-day-old S. torvum seedlings grown on MS-Gelrite plates were inoculated with 200–300 surface-sterilized J2s of A2-J or A2-O, or treated with SDW in vitro. Since there were clear morphological differences between the root tips infected with A2-J and A2-O after four days , it should follow that the success or failure of infection is determined within a few days post inoculation. We therefore decided to analyze the transcriptome at 1–3 DPI, corresponding to the early stages of infection. In addition, to detect gene expression in cells directly affected by the nematodes, we carefully collected infected root tips under a stereomicroscope . Root tips were cut with precision forceps and flash-frozen with liquid nitrogen to preclude the induction of wound responses. More than 50 root tips were pooled for each treatment, bucket flower and four biological replicates were used for the RNA-seq based transcriptome analyses.
We also carried out RNA-seq of whole roots and shoots of S. torvum infected with A2-J or A2-O, or mock treatment to improve the completeness of de novo transcriptome assembly. As a result, we obtained 218,024,788 paired-end reads from root tips and 341,297,551 single-end reads from whole shoots and roots after quality filtering . After removing the reads derived from nematodes, we performed de novo assembly using multiple assemblers with a variety of k-mer sizes . The final assembly had 88,596 contigs with an N50 of 1,298 bp, an average size of 800.62 bp, and a total length of 70,931,593 bp . We assessed the accuracy and completeness of the final assembly using BUSCO. The assembly included an estimated > 95 % of the assessed dataset, improving the current status of the transcriptome assembly of S. torvum and provided a high-quality transcriptome assembly of S. torvum for further analyses. Differential expression analysis showed that 1,220 genes were significantly up-regulated and 261 genes were down-regulated upon infection with A2-J, while 2,535 genes were up-regulated and 802 genes were down-regulated by infection with A2-O at at least one-time point during root tip infection, compared to the mock treatment . 1,029 genes were upregulated, and 180 genes were down-regulated at at least onetime point in both A2-J and A2-O infected plants .
Previous studies showed that the expression of genes associated with the salicylic acid , jasmonic acid , and ethylene signaling pathways are induced in resistant plants infected with PPNs , so we investigated the expression of marker genes for hormone biosynthesis, hormone signaling , and defense responses . Importantly, at 1 DPI, A2-J did not induce any statistically significant changes in the expression of any genes, whereas A2-O induced 204 genes, suggesting that infection with A2-O rapidly induces the expression of early responsive genes, which is prevented or avoided in A2-J infection. Since the speed of a defense response is one of the most important factors for successful immunity against pathogens, we hypothesized there must be important defense components among the 204 up-regulated genes. We therefore performed a GO enrichment analysis to identify significantly represented GO terms amongst the 204 up-regulated genes . The list of enriched GO terms was further reduced using “Reduce to most specific terms” option in Blast2GO to remove general GO terms and obtain only the most specific terms . Some GO terms that were significantly enriched among the 204 genes were related to the biosynthesis of isoprenoids ”, “sesquiterpene biosynthetic process ”, and “terpenoid biosynthetic process ”. To follow up on this result, we checked the expression of all the genes up-regulated by A2-O that are related to isoprenoid biosynthesis and found that A2-O infection induced the expression of genes encoding sesquiterpene synthases, such as viridiflorene synthase, vetispiradiene synthase, germacrene C synthase-like protein, and 5-epiaristolochene synthase.
Several other enzymes involved in isoprenoid biosynthesis, such as xanthoxin dehydrogenaselike protein and UDP-glycosyltransferase 91C1 were also up-regulated . Sesquiterpene synthases convert farnesyl diphosphate to sesquiterpenes such as germacrene C, 5-epiaristolochene, viridiflorene, and vetispiradiene. Because some isoprenoids have nematicidal activity , it is possible that the sesquiterpenes produced by S. torvum in response to infection with A2-O are nematicidal and contribute to suppressing A2-O infection. Other GO terms significantly enriched among the 204 up-regulated genes were related to oxidative stress ” and “response to oxidative stress ”. The most up-regulated genes by A2-Oin the GO term group were class III peroxidases, which are involved in lignification, cell elongation, seed germination, and response to abiotic and biotic stresses . The transcriptional up-regulation of the class III peroxidases is consistent with the fact that resistant tomato lines more strongly elevate peroxidase activity during RKN infection than susceptible lines . To identify the expression pattern of genes that are specific to A2-J or A2-O infection and common in both pathotypes, we clustered genes according to their transcript profiles by PCA with SOM clustering. SOM clustering grouped 6,502 genes into nine clusters based on their differential gene expression profiles after mock treatment or infection with either A2-J or A2-O . 429 genes in Cluster 2 and 554 genes in Cluster 4 were specifically up-regulated after infection with A2-J. In contrast, 1,769 and 600 genes in Cluster 8 and 9, respectively, were specifically up-regulated after infection with A2-O. 1,000 genes in Cluster 7 were up-regulated after infection with either A2-J or A2-O . We once again used GO enrichment to identify functional terms enriched in the genes in each cluster by further filtering enriched GO terms using the “Reduce to most specific terms” option in Blast2GO. In Cluster 4 , significantly enriched GO terms were related to cell wall remodeling, including “cell wall modification ”,“cell wall organization or biogenesis ”, and “pectin catabolic process ” . This is consistent with the observation that the expansion of giant cells is associated with an increase in cell wall thickness . GO terms associated with the significantly A2-J up-regulated genes include enzymes such as cellulose synthase-like protein, xyloglucan endotransglucosylase/hydrolase protein, and a non-catalytic subunit of a polygalacturonase isozyme . The transcriptional up-regulation of these enzymes is consistent with the presence of the common polysaccharides pectic homogalacturonan, xyloglucan, cut flower bucket and pectic arabinan in the cell walls of giant cells . Similarly, A2-J infection also activated the expression of COBRA-like protein, expansin, and LRR-RLK PXC1, which play important roles in cellulose deposition, loosening of cell walls, and secondary wall formation . Another GO term significantly enriched in Cluster 4 was “transmembrane transport ” . Other significantly up-regulated genes encode sugar transporter ERD6-like protein and amino acid transporter family protein . These transporters may promote the uptake of nutrients into giant cells or alter transportation through cells surrounding giant cells . We also performed GO enrichment analyses for Cluster 2, but no GO terms were enriched. In addition to GO enrichment analyses, we looked for interesting genes whose expression were dramatically upregulated in Clusters 2 and 4. These clusters included genes encoding chalcone synthase and a spermidine synthase that were specifically and highly expressed after infection with A2- J . Chalcone synthase is the first enzyme of the flavonoid biosynthetic pathway , and spermidine synthase is a key enzyme involved in polyamine biosynthesis . In summary, A2-J infection significantly and specifically up-regulates genes related to cell wall modification 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, 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 . These results suggest that the infection with A2-O transcriptionally activates lignin biosynthesis. 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 .