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

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

Journal of Nematology
Volume 53 (2021): Issue 1 (January 2021)
By: Lei Zhang and  Cynthia Gleason  
Open Access
|Oct 2021

Figures & Tables

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_

LibraryBio-replicateRead pairsRead pairs mapped to genomeMapping rate (%)
Pre-parasitic J2A51 950 81525 570 65949.60%
Pre-parasitic J2B43 883 14321 477 89249.00%
Pre-parasitic J2C44 867 52922 072 54549.20%
Mc1-potato 8 dpiA46 115 0171 572 4043.40%
Mc1-potato 8 dpiB40 726 1721 294 7723.20%
Mc1-potato 8 dpiC40 068 6461 357 7593.40%

Information about the 11 M_ chitwoodi genes analyzed by qRT-PCR at different life stages_

Gene ID (aa)BLASTx nrBLASTx % query coverBLASTx e-valuePredicted domainsFPKM J2FPKM 8 dpi
Mc07118 (171) Meloidogyne graminicola MSP18 mRNA, complete cds, Genbank: MK628546.1847.00E-40N/A273.9
Mc10102 (103)Unnamed protein product [Meloidogyne enterolobii] Genbank: CAD2180454.1794.00E-46Cytochrome b5, heme binding domain16.7418.6
Mc08299 (207)Cathepsin B [Meloidogyne graminicola]Genbank KAF7630769.1897.00E-45cysteine proteinase26.4476
Mc04921 (544)hypothetical protein Mgra_00005223 [Meloidogyne graminicola] Genbank KAF7635403.1346.00E-74Thioredoxin-like fold2.950.4
Mc06869 (63)N/A N/A39.21704.9
Mc03992 (306)Hypothetical protein Mgra_00005223 [Meloidogyne graminicola] Genbank KAF76334147.1493.20E-02N/A4.369.8
Mc01565 (430)Unnamed protein product [Meloidogyne enterolobii] Genbank: CAD2124200.1182.00E-22N/A7.3288.1
Mc08895 (332)Unnamed protein product [Meloidogyne enterolobii] Genbank: CAD2174631.1325.00E-25N/A3.5123.7
Mc04677 (315)Hypothetical protein Mgra_00005223 [Meloidogyne graminicola] Genbank KAF7635986.1597.00E-54N/A2.351.8
Mc03439 (79)Hypothetical protein Mgra_00005223 [Meloidogyne graminicola] Genbank KAF7630992.1971.00E-23N/A25.81319.5
Mc10400 (130)N/A N/A7.2154.8

Top 10 most differentially expressed genes in parasitic M_ chitwoodi juveniles_

Log FCDescription
Up-regulated112.2878Putative cuticular collagen
210.8863---NA---
310.6047Putative esophageal gland cell secretory protein 14
410.5679---NA---
510.5294Hydroxyacyl-coenzyme A dehydrogenase, mitochondrial
69.2692---NA---
79.21531---NA---
89.21423Nematode cuticle collagen domain protein
99.1679Saposin-like type B, 1 domain and Saposin B domain and Saposin-like domain-containing protein
109.0144---NA---
Down-regulated1-11.012C-type lectin domain-containing protein
2-10.711Mucin-like protein-1
3-10.681SCP domain-containing protein
4-10.443---NA---
5-10.308Beta-1,4-endoglucanase
6-10.234C-type lectin domain-containing protein
7-10.205Mucin-like protein-1
8-9.9796---NA---
9-9.975---NA---
10-9.8201Hypothetical protein Mgra_00005110, partial

Primers for qRT-PCR_

Name5′-3′
McITS2 forwardGGGGTCAAACCCTTTGGCACGTCTGG
McITS2 reverseGCGGGTGATCTCGACTGAGTTCAGG
Mc00773 forwardAAGTTGCCGATAGCATTGCG
Mc00773 reverseTCCGGAAGAGCATGACGAAT
Mc03439 forwardTGCAGTTGCTGAGAGTTGTC
Mc03439 reverseTGCATGGTGGAATAACCAAAGT
Mc03992 forwardTGATCGTTCGACTTCTGGACA
Mc03992 reverseGCACGAGTGCCTTGAACTTG
Mc04677 forwardCTGCACATCAGAAGAAAAATGCT
Mc04677 reverseTCCACAGGGTCACAACTTCC
Mc04921 forwardTCGTGTTAGCGGTGATGGTT
Mc04921 reverseCGTTTCGGTGCCAAGTTCAG
Mc08895 forwardTCTTTGCACGAAGTTGATCG
Mc08895 reverseTTTAATCCATTTACAAAAAGCCCT
Mc10400 forwardCAAGGAGGTGGAAAAACGCC
Mc10400 reverseGGGTTCTTGATTCCCAACCG
Mc01565 forwardCAGCTGAATCTTCTGCCCCA
Mc01565 reverseAATAGCAACGGCTGGAGCTT
Mc06869 forwardCCACATCATCATCATCATTCAAGT
Mc06869 reverseCCCATGGCCAAGTTGAACC
Mc08299 forwardGAATTACGCCGCTTTCTTGGT
Mc08299 reverseTGCACATTCAGGCCACTCAT
Mc07118 forwardTGGTTGTTATGAACATTCTGGCG
Mc07118 reverseCCAGGTTTGTTAGGTAAGGCAG
Mc10102 forwardTCCACATCATCCTGGAGGTC
Mc10102 reverseATGAAAAGCACCAGCCCCAT
DOI: https://doi.org/10.21307/jofnem-2021-084 | Journal eISSN: 2640-396X | Journal ISSN: 0022-300X
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Language: English
Page range: 1 - 13
Published on: Oct 12, 2021
Published by: Society of Nematologists, Inc.
In partnership with: Paradigm Publishing Services
Publication frequency: 1 issue per year
Keywords:
Differentially expressed genes, Host-parasitic relationship,
Meloidogyne,
Molecular biology,
Transcriptome
Related subjects:
Life sciences,
Life sciences, other

© 2021 Lei Zhang, Cynthia Gleason, published by Society of Nematologists, Inc.
This work is licensed under the Creative Commons Attribution 4.0 License.

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