Direct identification of the Meloidogyne incognita secretome reveals proteins with host cell reprogramming potential

Abstract

The root knot nematode, Meloidogyne incognita, is an obligate parasite that causes significant damage to a broad range of host plants. Infection is associated with secretion of proteins surrounded by proliferating cells. Many parasites are known to secrete effectors that interfere with plant innate immunity, enabling infection to occur; they can also release pathogen-associated molecular patterns (PAMPs, e.g., flagellin) that trigger basal immunity through the nematode stylet into the plant cell. This leads to suppression of innate immunity and reprogramming of plant cells to form a feeding structure containing multinucleate giant cells. Effectors have generally been discovered using genetics or bioinformatics, but M. incognita is non-sexual and its genome sequence has not yet been reported. To partially overcome these limitations, we have used mass spectrometry to directly identify 486 proteins secreted by M. incognita. These proteins contain at least segmental sequence identity to those found in our 3 reference databases (published nematode proteins; unpublished M. incognita ESTs; published plant proteins). Several secreted proteins are homologous to plant proteins, which they may mimic, and they contain domains that suggest known effector functions (e.g., regulating the plant cell cycle or growth). Others have regulatory domains that could reprogram cells. Using in situ hybridization we observed that most secreted proteins were produced by the subventral glands, but we found that phasmids also secreted proteins. We annotated the functions of the secreted proteins and classified them according to roles they may play in the development of root knot disease. Our results show that parasite secretomes can be partially characterized without cognate genomic DNA sequence. We observed that the M. incognita secretome overlaps the reported secretome of mammalian parasitic nematodes (e.g., Brugia malayi), suggesting a common parasitic behavior and a possible conservation of function between metazoan parasites of plants and animals.

Figure 1. Sample process flow chart.

M. incognita proteins were collected from the aqueous medium (the secretome) and from extracts of worms. Proteins in the tomato root exudates (TRE) that diffused across a 3,500Da filter membrane were also collected. Proteins were identified by nanoLC ESI MS/MS. A, B, C and D are Protein databases used for protein identification, complementary information for each database are available on Table S3.

Figure 2. Distribution of secreted proteins identified in the protein databases.

Venn diagram showing the distribution of secreted proteins identified using the M. incognita, Other Nematode, or Plant protein databases. A complete description of all databases is shown in Table S3.

Figure 3. Relative abundance of secreted proteins compared to extracts from intact nematodes.

Spectrum counting was used for relative protein quantification compating the secretome to the intact nematode proteome. The number of valid MS/MS spectra from each protein was normalized to the total MS/MS spectra number of each dataset. The normalized spectrum count ratio of each protein (secretome/whole worm proteome) was used to evaluate if the protein was enriched in the secretome. Circle represents all secreted proteins identified in this study; Triangle represents proteins previously reported to be in the secretome (A complete description of these proteins is shown in Table S5); Diamond represents proteins that we found in M. incognita secretome and that could play a crucial role in the establishment of the host-pathogen compatibility. These proteins are discussed in detail.

Figure 4. Localization of gene expression by in situ hybridization.

Digoxigenin-labeled antisense cDNA probes of selected gene clones were hybridized to transcripts expressed within cells of J2 stage Meloidogyne incognita. Sections of the nematode were incubated with antisense probes designed based on DNA sequence of the following contigs: A, CL1191Contig1_1; B, CL312Contig1_1; C, CL5Contig2_1; D AF100549 (β-1,4-endoglucanase), E, CL2552Contig1_1; F, CL321Contig1_1; G, AF402771 (calreticulin); H, CL480Contig2_1. M = metacorpus, SvG = subventral glands.