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Transcriptome Analyses of Pre-parasitic and Parasitic Meloidogyne Chitwoodi Race 1 to Identify Putative Effector Genes

Lei Zhang
Lei Zhang
 and 
Cynthia Gleason
Cynthia Gleason
   | Oct 12, 2021
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Published Online: Oct 12, 2021
Page range: 1 - 13
DOI: https://doi.org/10.21307/jofnem-2021-084
Keywords
© 2021 Lei Zhang et al., published by Sciendo.
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Figure S1:

Modified Baermann funnel apparatus diagram. The modified Baermann funnel apparatus uses a 100 mL beaker instead of the traditional funnel. Mesh is placed into the mouth of the beaker and up to 4 layers of Kimwipes are placed on the mesh. Nematode eggs (or soil) are placed onto the paper and immersed in water. After three days, the paper and mesh are removed and the nematodes sitting at the bottom of the beaker are collected. The modified apparatus allows for J2 collection from small volumes of soil or isolated eggs.
Modified Baermann funnel apparatus diagram. The modified Baermann funnel apparatus uses a 100 mL beaker instead of the traditional funnel. Mesh is placed into the mouth of the beaker and up to 4 layers of Kimwipes are placed on the mesh. Nematode eggs (or soil) are placed onto the paper and immersed in water. After three days, the paper and mesh are removed and the nematodes sitting at the bottom of the beaker are collected. The modified apparatus allows for J2 collection from small volumes of soil or isolated eggs.

Figure 1:

Acid fuchsin staining of Meloidogyne chitwoodi in potato roots at 4 (A), 8 (B) and 16 (C) days post inoculation (dpi). Bars, 50 µm.
Acid fuchsin staining of Meloidogyne chitwoodi in potato roots at 4 (A), 8 (B) and 16 (C) days post inoculation (dpi). Bars, 50 µm.

Figure 2:

Gene Ontology (GO) term level 2 categories of genes for up (A) and down (B) regulated genes from M. chitwoodi parasitic stage compared to pre-parasitic J2. Bars show the number of sequences with annotations under each term. BP = biological processes, MP = molecular processes, CC = cellular function.
Gene Ontology (GO) term level 2 categories of genes for up (A) and down (B) regulated genes from M. chitwoodi parasitic stage compared to pre-parasitic J2. Bars show the number of sequences with annotations under each term. BP = biological processes, MP = molecular processes, CC = cellular function.

Figure 3:

Flowchart summary of RNA-seq analysis and prediction of Meloidogyne chitwoodi genes involved in parasitism. Bioinformatic and other tools are boxed. FC, fold change. FPKM, fragments per kilobase of transcript per million mapped reads.
Flowchart summary of RNA-seq analysis and prediction of Meloidogyne chitwoodi genes involved in parasitism. Bioinformatic and other tools are boxed. FC, fold change. FPKM, fragments per kilobase of transcript per million mapped reads.

Figure 4:

The expression level of selected M. chitwoodi genes in J2 and in parasitic nematodes in potato roots at 4, 8, and 16 dpi. McITS2 was used as the reference gene. The results show the fold change relative the juvenile stage, n = 3 ± SEM, * P < 0.05 using two-tailed Student’s t test.
The expression level of selected M. chitwoodi genes in J2 and in parasitic nematodes in potato roots at 4, 8, and 16 dpi. McITS2 was used as the reference gene. The results show the fold change relative the juvenile stage, n = 3 ± SEM, * P < 0.05 using two-tailed Student’s t test.

Meloidogyne chitwoodi RNA-seq libraries and mapping to genome.

Library Bio-replicate Read pairs Read pairs mapped to genome Mapping rate (%)
Pre-parasitic J2 A 51 950 815 25 570 659 49.60%
Pre-parasitic J2 B 43 883 143 21 477 892 49.00%
Pre-parasitic J2 C 44 867 529 22 072 545 49.20%
Mc1-potato 8 dpi A 46 115 017 1 572 404 3.40%
Mc1-potato 8 dpi B 40 726 172 1 294 772 3.20%
Mc1-potato 8 dpi C 40 068 646 1 357 759 3.40%

Information about the 11 M. chitwoodi genes analyzed by qRT-PCR at different life stages.

Gene ID (aa) BLASTx nr BLASTx % query cover BLASTx e-value Predicted domains FPKM J2 FPKM 8 dpi
Mc07118 (171) Meloidogyne graminicola MSP18 mRNA, complete cds, Genbank: MK628546.1 84 7.00E-40 N/A 2 73.9
Mc10102 (103) Unnamed protein product [Meloidogyne enterolobii] Genbank: CAD2180454.1 79 4.00E-46 Cytochrome b5, heme binding domain 16.7 418.6
Mc08299 (207) Cathepsin B [Meloidogyne graminicola]Genbank KAF7630769.1 89 7.00E-45 cysteine proteinase 26.4 476
Mc04921 (544) hypothetical protein Mgra_00005223 [Meloidogyne graminicola] Genbank KAF7635403.1 34 6.00E-74 Thioredoxin-like fold 2.9 50.4
Mc06869 (63) N/A N/A 39.2 1704.9
Mc03992 (306) Hypothetical protein Mgra_00005223 [Meloidogyne graminicola] Genbank KAF76334147.1 49 3.20E-02 N/A 4.3 69.8
Mc01565 (430) Unnamed protein product [Meloidogyne enterolobii] Genbank: CAD2124200.1 18 2.00E-22 N/A 7.3 288.1
Mc08895 (332) Unnamed protein product [Meloidogyne enterolobii] Genbank: CAD2174631.1 32 5.00E-25 N/A 3.5 123.7
Mc04677 (315) Hypothetical protein Mgra_00005223 [Meloidogyne graminicola] Genbank KAF7635986.1 59 7.00E-54 N/A 2.3 51.8
Mc03439 (79) Hypothetical protein Mgra_00005223 [Meloidogyne graminicola] Genbank KAF7630992.1 97 1.00E-23 N/A 25.8 1319.5
Mc10400 (130) N/A N/A 7.2 154.8

Top 10 most differentially expressed genes in parasitic M. chitwoodi juveniles.

Log FC Description
Up-regulated 1 12.2878 Putative cuticular collagen
2 10.8863 ---NA---
3 10.6047 Putative esophageal gland cell secretory protein 14
4 10.5679 ---NA---
5 10.5294 Hydroxyacyl-coenzyme A dehydrogenase, mitochondrial
6 9.2692 ---NA---
7 9.21531 ---NA---
8 9.21423 Nematode cuticle collagen domain protein
9 9.1679 Saposin-like type B, 1 domain and Saposin B domain and Saposin-like domain-containing protein
10 9.0144 ---NA---
Down-regulated 1 -11.012 C-type lectin domain-containing protein
2 -10.711 Mucin-like protein-1
3 -10.681 SCP domain-containing protein
4 -10.443 ---NA---
5 -10.308 Beta-1,4-endoglucanase
6 -10.234 C-type lectin domain-containing protein
7 -10.205 Mucin-like protein-1
8 -9.9796 ---NA---
9 -9.975 ---NA---
10 -9.8201 Hypothetical protein Mgra_00005110, partial

Primers for qRT-PCR.

Name 5′-3′
McITS2 forward GGGGTCAAACCCTTTGGCACGTCTGG
McITS2 reverse GCGGGTGATCTCGACTGAGTTCAGG
Mc00773 forward AAGTTGCCGATAGCATTGCG
Mc00773 reverse TCCGGAAGAGCATGACGAAT
Mc03439 forward TGCAGTTGCTGAGAGTTGTC
Mc03439 reverse TGCATGGTGGAATAACCAAAGT
Mc03992 forward TGATCGTTCGACTTCTGGACA
Mc03992 reverse GCACGAGTGCCTTGAACTTG
Mc04677 forward CTGCACATCAGAAGAAAAATGCT
Mc04677 reverse TCCACAGGGTCACAACTTCC
Mc04921 forward TCGTGTTAGCGGTGATGGTT
Mc04921 reverse CGTTTCGGTGCCAAGTTCAG
Mc08895 forward TCTTTGCACGAAGTTGATCG
Mc08895 reverse TTTAATCCATTTACAAAAAGCCCT
Mc10400 forward CAAGGAGGTGGAAAAACGCC
Mc10400 reverse GGGTTCTTGATTCCCAACCG
Mc01565 forward CAGCTGAATCTTCTGCCCCA
Mc01565 reverse AATAGCAACGGCTGGAGCTT
Mc06869 forward CCACATCATCATCATCATTCAAGT
Mc06869 reverse CCCATGGCCAAGTTGAACC
Mc08299 forward GAATTACGCCGCTTTCTTGGT
Mc08299 reverse TGCACATTCAGGCCACTCAT
Mc07118 forward TGGTTGTTATGAACATTCTGGCG
Mc07118 reverse CCAGGTTTGTTAGGTAAGGCAG
Mc10102 forward TCCACATCATCCTGGAGGTC
Mc10102 reverse ATGAAAAGCACCAGCCCCAT
eISSN:
2640-396X
Language:
English
Publication timeframe:
Volume Open
Journal Subjects:
Life Sciences, other
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