Identification of genes involved in Meloidogyne incognita-induced gall formation processes in Arabidopsis thaliana

Abstract

Root-knot nematodes (RKN; Meloidogyne incognita) are phytoparasitic nematodes that cause significant damage to crop plants worldwide. Recent studies have revealed that RKNs disrupt various physiological processes in host plant cells to induce gall formation. However, little is known about the molecular mechanisms of gall formation induced by nematodes. We have previously found that RNA expression levels of some of genes related to micro-RNA, cell division, membrane traffic, vascular formation, and meristem maintenance system were modified by nematode infection. Here we evaluated these genes importance during nematode infection by using Arabidopsis mutants and/or β-glucronidase (GUS) marker genes, particularly after inoculation with nematodes, to identify the genes involved in successful nematode infection. Our results provide new insights not only for the basic biology of plant-nematode interactions but also to improve nematode control in an agricultural setting.

Keywords: gall formation; plant–microorganism interaction; root-knot nematodes.

Figure 1. Effect of mutations in cell cycle-associated genes during M. incognita infection. Quantitative analysis of gall numbers at 14 dpi (A). Note that the comparison of gall numbers between the wild-type and the mutants was performed independently. (p**<0.01; p*<0.05) Means±SD are shown. p-values were analyzed by Student’s t-test. Promoter activities of cell cycle-associated genes were induced in galls. Five-day-old Arabidopsis seedlings harboring the CYCB1;1::GUS, MYB3R4::GUS, or MYB3R5::GUS constructs were inoculated with M. incognita (B). GUS activity was detected in the vascular bundles or lateral root primordium (LRP) of non-infected (NI) roots, while GUS signals were expressed in galls 7 dpi. Activities of CDKB1;1::CDKB1;1-GUS and CDKB1;2::CDKB2;1-GUS were induced in galls. Five-day-old Arabidopsis seedlings harboring the CDKB1;1::CDKB1;1-GUS or CDKB2;1::CDKB2;1-GUS constructs were inoculated with M. incognita. (C) GUS activity was detected in the LRP and root tips (no date) of NI roots, while GUS signals were stronger in galls on 1, 2, 3, 5, and 7 dpi. Scale bar=100 µm; dpi=days post-infection.

Figure 2. Effect of mutations in miRNA-associated genes during M. incognita infection. Quantitative analysis of the number of galls at 14 dpi. Note that the comparison of gall numbers between the wild-type and the mutants was performed independently. (p**<0.01, p*<0.05) Means±SD are shown. p-values were analyzed by Student’s t-test, with the exception of ago7-1, for which the U test was used.

Figure 3. Effect of mutations in membrane transport-associated genes during M. incognita infection. Quantitative analysis of the gall numbers at 14 dpi (A). Note that the comparison of gall numbers between the wild-type and the mutants was performed independently. Means±SD are shown. p-values were analyzed by Student’s t-test.

Figure 4. Promoter activities of SAM-associated genes are induced in galls. Five-day-old Arabidopsis seedlings harboring the CUC2::GUS, KNAT1::GUS, BAM1::GUS (Col-0 background) or STM::GUS (Ler background) constructs were inoculated with M. incognita (B). GUS activity was detected in the stems of NI roots, while GUS signals were expressed within the vascular cylinder in galls 7 dpi. Scale bar=100 µm; SAM=shoot apical meristem.

Figure 5. Promoter activities of vascular-associated genes are induced in galls. Five-day-old Arabidopsis seedlings harboring the AHA3::GUS, APL::GUS, SUC2::GUS or VH1::GUS constructs were inoculated with M. incognita (B). GUS activity was detected in the vascular bundles of NI roots, while GUS signals were expressed within the vascular cylinder in galls 7 dpi. Scale bar=100 µm.