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Solanum torvum responses to the root-knot nematode Meloidogyne incognita.

Bagnaresi P, Sala T, Irdani T, Scotto C, Lamontanara A, Beretta M, Rotino GL, Sestili S, Cattivelli L, Sabatini E - BMC Genomics (2013)

Bottom Line: GO term enrichment analyses with the 390 Torvum DEG revealed enhancement of several processes as chitin catabolism and sesquiterpenoids biosynthesis, while no GO term enrichment was found with eggplant DEG.The genes identified from S. torvum catalogue, bearing high similarity to known nematode resistance genes, were further investigated in view of their potential role in the nematode resistance mechanism.By combining 454 pyrosequencing and microarray technology we were able to conduct a cost-effective global transcriptome profiling in a non-model species.The expression profiling of S. torvum responses to nematode infection points to sesquiterpenoids and chitinases as major effectors of nematode resistance.

View Article: PubMed Central - HTML - PubMed

Affiliation: Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Genomics Research Centre, via S Protaso 302, I-29107 Fiorenzuola d’Arda, PC, Italy.

ABSTRACT

Background: Solanum torvum Sw is worldwide employed as rootstock for eggplant cultivation because of its vigour and resistance/tolerance to the most serious soil-borne diseases as bacterial, fungal wilts and root-knot nematodes. The little information on Solanum torvum (hereafter Torvum) resistance mechanisms, is mostly attributable to the lack of genomic tools (e.g. dedicated microarray) as well as to the paucity of database information limiting high-throughput expression studies in Torvum.

Results: As a first step towards transcriptome profiling of Torvum inoculated with the nematode M. incognita, we built a Torvum 3' transcript catalogue. One-quarter of a 454 full run resulted in 205,591 quality-filtered reads. De novo assembly yielded 24,922 contigs and 11,875 singletons. Similarity searches of the S. torvum transcript tags catalogue produced 12,344 annotations. A 30,0000 features custom combimatrix chip was then designed and microarray hybridizations were conducted for both control and 14 dpi (day post inoculation) with Meloidogyne incognita-infected roots samples resulting in 390 differentially expressed genes (DEG). We also tested the chip with samples from the phylogenetically-related nematode-susceptible eggplant species Solanum melongena. An in-silico validation strategy was developed based on assessment of sequence similarity among Torvum probes and eggplant expressed sequences available in public repositories. GO term enrichment analyses with the 390 Torvum DEG revealed enhancement of several processes as chitin catabolism and sesquiterpenoids biosynthesis, while no GO term enrichment was found with eggplant DEG.The genes identified from S. torvum catalogue, bearing high similarity to known nematode resistance genes, were further investigated in view of their potential role in the nematode resistance mechanism.

Conclusions: By combining 454 pyrosequencing and microarray technology we were able to conduct a cost-effective global transcriptome profiling in a non-model species. In addition, the development of an in silico validation strategy allowed to further extend the use of the custom chip to a related species and to assess by comparison the expression of selected genes without major concerns of artifacts. The expression profiling of S. torvum responses to nematode infection points to sesquiterpenoids and chitinases as major effectors of nematode resistance. The availability of the long sequence tags in S. torvum catalogue will allow precise identification of active nematocide/nematostatic compounds and associated enzymes posing the basis for exploitation of these resistance mechanisms in other species.

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Explanation of probe filtering metrics for eggplant transcripts. Parameters used for filtering of Torvum probes based on homology to eggplant transcript are shown. Due to the steric constraints in the crowded 3’ attachment to chip region, the 3′ region of probes is less accessible and therefore mismatches in the region are less influential.
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Figure 3: Explanation of probe filtering metrics for eggplant transcripts. Parameters used for filtering of Torvum probes based on homology to eggplant transcript are shown. Due to the steric constraints in the crowded 3’ attachment to chip region, the 3′ region of probes is less accessible and therefore mismatches in the region are less influential.

Mentions: To validate the heterologous expression data, we followed an approach conceptually similar to that presented in Bagnaresi et al. [27]. Toward this end, we first pooled several expression (EST and assembled unigenes) eggplant databases and queried the merged eggplant database with Torvum chip probe sequences using local BlastN at a relaxed stringency. Alignment results were parsed to filter probes based on alignment parameters expected to influence hybridization strength. The following parameters were considered (see explanatory Figure 3): i) ratio of alignment length to oligo length (percent alignment); ii) maximum number of mismatches; and iii) distance from the start of oligo/transcript alignment to oligo 5’ end. The rationale for the choice of these parameters is based on data presented in Additional file 8. The mean of all expression values (both control and infected conditions, Additional file 8, panel A) for all 23,284 probes (i.e. no filtering) was 945 (727 when considering only probes with an alignment <= 40%; data not shown). When probes are selected by filtering with the above parameters, the mean of all expression values reaches the maximum for probes filtered for percent alignment = 100% and 0 mismatches (808 probes conserved; mean hybridization value 2,424) (Additional file 8, panel A). On the other hand, when less stringent values were tested, down to the very relaxed >= 30% alignment and up to 3 mismatches, mean expression values drop to about 900 (23,284 probes conserved) (Additional file 8, panel A). This variation in expression values is consistent with the fact that, when the homology between chip oligos and eggplant transcripts is high, high hybridization values are detectable. Additionally, the influence of the distance between the starting point of oligo alignment and the respective Blast hit within 5′ end was monitored (Additional file 8, panels B to E). In fact, oligos are attached to the chip in the 3’ side and this causes steric hindrance in the crowded 3’ regions to interfere with hybridization [30]. Therefore, poor homology along oligo 3’ region against interrogating transcripts can be expected to be less influential on hybridization intensity, as confirmed in our data No effect could be detected when selection was made for distance of alignment from 3’ side (data not shown). We further checked that the variation in mean hybridization is not a mere consequence of the varying number of probes filtered at the different imposed stringencies. A plot where a number of random probes corresponding to the number of probes resulting by setting stringency conditions is shown in Additional file 8F. As expected, no meaningful variation in signal strength is detectable in this case, ruling out that simply the number of probes, irrespective of probe vs. interrogating sequence homology, is influential. All these observations are summarized in Additional file 8, where percent alignment, number of mismatches within the alignment and oligo alignment start position are plotted versus mean hybridization values and number of retained probes.


Solanum torvum responses to the root-knot nematode Meloidogyne incognita.

Bagnaresi P, Sala T, Irdani T, Scotto C, Lamontanara A, Beretta M, Rotino GL, Sestili S, Cattivelli L, Sabatini E - BMC Genomics (2013)

Explanation of probe filtering metrics for eggplant transcripts. Parameters used for filtering of Torvum probes based on homology to eggplant transcript are shown. Due to the steric constraints in the crowded 3’ attachment to chip region, the 3′ region of probes is less accessible and therefore mismatches in the region are less influential.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750854&req=5

Figure 3: Explanation of probe filtering metrics for eggplant transcripts. Parameters used for filtering of Torvum probes based on homology to eggplant transcript are shown. Due to the steric constraints in the crowded 3’ attachment to chip region, the 3′ region of probes is less accessible and therefore mismatches in the region are less influential.
Mentions: To validate the heterologous expression data, we followed an approach conceptually similar to that presented in Bagnaresi et al. [27]. Toward this end, we first pooled several expression (EST and assembled unigenes) eggplant databases and queried the merged eggplant database with Torvum chip probe sequences using local BlastN at a relaxed stringency. Alignment results were parsed to filter probes based on alignment parameters expected to influence hybridization strength. The following parameters were considered (see explanatory Figure 3): i) ratio of alignment length to oligo length (percent alignment); ii) maximum number of mismatches; and iii) distance from the start of oligo/transcript alignment to oligo 5’ end. The rationale for the choice of these parameters is based on data presented in Additional file 8. The mean of all expression values (both control and infected conditions, Additional file 8, panel A) for all 23,284 probes (i.e. no filtering) was 945 (727 when considering only probes with an alignment <= 40%; data not shown). When probes are selected by filtering with the above parameters, the mean of all expression values reaches the maximum for probes filtered for percent alignment = 100% and 0 mismatches (808 probes conserved; mean hybridization value 2,424) (Additional file 8, panel A). On the other hand, when less stringent values were tested, down to the very relaxed >= 30% alignment and up to 3 mismatches, mean expression values drop to about 900 (23,284 probes conserved) (Additional file 8, panel A). This variation in expression values is consistent with the fact that, when the homology between chip oligos and eggplant transcripts is high, high hybridization values are detectable. Additionally, the influence of the distance between the starting point of oligo alignment and the respective Blast hit within 5′ end was monitored (Additional file 8, panels B to E). In fact, oligos are attached to the chip in the 3’ side and this causes steric hindrance in the crowded 3’ regions to interfere with hybridization [30]. Therefore, poor homology along oligo 3’ region against interrogating transcripts can be expected to be less influential on hybridization intensity, as confirmed in our data No effect could be detected when selection was made for distance of alignment from 3’ side (data not shown). We further checked that the variation in mean hybridization is not a mere consequence of the varying number of probes filtered at the different imposed stringencies. A plot where a number of random probes corresponding to the number of probes resulting by setting stringency conditions is shown in Additional file 8F. As expected, no meaningful variation in signal strength is detectable in this case, ruling out that simply the number of probes, irrespective of probe vs. interrogating sequence homology, is influential. All these observations are summarized in Additional file 8, where percent alignment, number of mismatches within the alignment and oligo alignment start position are plotted versus mean hybridization values and number of retained probes.

Bottom Line: GO term enrichment analyses with the 390 Torvum DEG revealed enhancement of several processes as chitin catabolism and sesquiterpenoids biosynthesis, while no GO term enrichment was found with eggplant DEG.The genes identified from S. torvum catalogue, bearing high similarity to known nematode resistance genes, were further investigated in view of their potential role in the nematode resistance mechanism.By combining 454 pyrosequencing and microarray technology we were able to conduct a cost-effective global transcriptome profiling in a non-model species.The expression profiling of S. torvum responses to nematode infection points to sesquiterpenoids and chitinases as major effectors of nematode resistance.

View Article: PubMed Central - HTML - PubMed

Affiliation: Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Genomics Research Centre, via S Protaso 302, I-29107 Fiorenzuola d’Arda, PC, Italy.

ABSTRACT

Background: Solanum torvum Sw is worldwide employed as rootstock for eggplant cultivation because of its vigour and resistance/tolerance to the most serious soil-borne diseases as bacterial, fungal wilts and root-knot nematodes. The little information on Solanum torvum (hereafter Torvum) resistance mechanisms, is mostly attributable to the lack of genomic tools (e.g. dedicated microarray) as well as to the paucity of database information limiting high-throughput expression studies in Torvum.

Results: As a first step towards transcriptome profiling of Torvum inoculated with the nematode M. incognita, we built a Torvum 3' transcript catalogue. One-quarter of a 454 full run resulted in 205,591 quality-filtered reads. De novo assembly yielded 24,922 contigs and 11,875 singletons. Similarity searches of the S. torvum transcript tags catalogue produced 12,344 annotations. A 30,0000 features custom combimatrix chip was then designed and microarray hybridizations were conducted for both control and 14 dpi (day post inoculation) with Meloidogyne incognita-infected roots samples resulting in 390 differentially expressed genes (DEG). We also tested the chip with samples from the phylogenetically-related nematode-susceptible eggplant species Solanum melongena. An in-silico validation strategy was developed based on assessment of sequence similarity among Torvum probes and eggplant expressed sequences available in public repositories. GO term enrichment analyses with the 390 Torvum DEG revealed enhancement of several processes as chitin catabolism and sesquiterpenoids biosynthesis, while no GO term enrichment was found with eggplant DEG.The genes identified from S. torvum catalogue, bearing high similarity to known nematode resistance genes, were further investigated in view of their potential role in the nematode resistance mechanism.

Conclusions: By combining 454 pyrosequencing and microarray technology we were able to conduct a cost-effective global transcriptome profiling in a non-model species. In addition, the development of an in silico validation strategy allowed to further extend the use of the custom chip to a related species and to assess by comparison the expression of selected genes without major concerns of artifacts. The expression profiling of S. torvum responses to nematode infection points to sesquiterpenoids and chitinases as major effectors of nematode resistance. The availability of the long sequence tags in S. torvum catalogue will allow precise identification of active nematocide/nematostatic compounds and associated enzymes posing the basis for exploitation of these resistance mechanisms in other species.

Show MeSH
Related in: MedlinePlus