| Root-knot nematode, an important kind of plant parasitic nematodes, causes huge economic losses to agricultural production because of its extensive host range and serious damage. The second-stage juveniles of root-knot nematode infect root of host plants, then inject secreted proteins of esophageal gland through stylet, which induce the formation of giant cells differentiated by vascular tissue cells of host plant and thus root knots,meanwhile absorb nutrition from host plants constantly by their stylets, so that make a very serious influence to the normally growth and development of host plants. So far, the control of the root-knot nematodes is still in the traditional method, which makes it quite difficult.With the development of molecular biology, research on the molecular interaction mechanism of root-knot nematode and giant cells of host plants by molecular biology technology deeply, cultivating new species of transgenic anti-nematode plants, cutting off the infection way of root-knot nematode fundamentally, and getting effective control of the root-knot nematode, has became a new direction. So it is extremely important to understand the development of giant cells. Some research found that giant cells showed obvious vacuolation and less inclusions volume in the later stage but the specific process of vacuolation is still not clear. Ipomoea aquatica is an ideal host plant of the root-knot nematodes. In this paper, we make I. Aquatica as the host plant to study their giant cells,which lays a foundation of revealing the vacuolation process of giant cells caused by root-knot nematodes. Main innovative results are as follows:1. Paraffin section technology was optimized. According to the characteristic of young tissue and moisture content of root-knot from I. aquatica roots, whole staining of root-knot was applied instead of traditional staining after lost-wax, meanwhile crosscutting also was used. In the resulting picture, root-knot tissue had a good staining and clear structure, especially for giant cells, root-knot nematode and cell nucleus.2. The optimized paraffin section technology was used to study the development process of giant cells and its inclusions in I. aquatica root knot firstly. The area and diameter of giant cells and its inclusions had the same developmental trend. Firstly, they increased and reached their maximum size on 27 th DPI(days post inoculation), area were26917.4±314.7 ?m2 and 22302.5±376.2 ?m2 respectively and diameter were 276.5±6.2?m and 254.5±7.7 ?m respectively. Secondly, they decreased to their minimum size on32 th DPI, area were 8677.9±170.6 ?m2 and 7902.7±113.0 ?m2 respectively and diameter were 185.5±5.1 ?m and 151.0±6.5 ?m respectively. Finally they increased again and reached their maximum size on 43 th DPI, area were 28319.8±331.9 ?m2 and 18619.3±331.2 ?m2 respectively and diameter were 393.0±5.5 ?m and 332.5±3.1 ?m respectively.The number of nuclei increased firstly and reached its maximum on 23 th DPI, which was27.6±2.2. Then it decreased to minimum on 32 th DPI, which was 9.8±0.9. Finally it increased again and became maximum on 43 th DPI, which was 29±1.0. The area of nuclei became maximum on 32 th DPI, which was 163.7±4.6 ?m2, then it decreased to minimum on 41 th DPI, which was 106.2±2.9 ?m2.3. The characteristic of apoptotic giant cells of I. aquatica root knot was revealed with shrinking giant cells, thickening cell wall, sparse inclusion, intumescent and lessening nucleus. Positive green fluorescence signals in the giant cells of the 32 DPI was detected by optimized TUNEL at the single cell level, while no green fluorescence signals were detected in the negative control. These results showed the apoptosis of giant cells in the later stage.4. Mi PDCD6 proteins of M. incognita were localized in I. aquatica root knot by immunofluorescence localization technology. Root knots collected after 8 days, 31 days and 32 days of M. incognita infestation were used for paraffin sections. Then immunofluorescence localization of the Mi PDCD6 proteins from M. incognita in the root-knot tissues of host plant was conducted. Results were observed and photographed by ZEISS LSCM 780 confocal laser scanning microscope.There were some green fluorescence signals in the giant cells of 8 DPI, and some stronger green fluorescence signals in the giant cells of 31 DPI and 32 DPI. Stronger green fluorescence signals were also detected around the stylet and in the body of M. incognita. These results demonstrated that the Mi PDCD6 proteins did express in M. incognita, and had been secreted into the giant cells of their host plant.5. The histopathological structure of root-knot after the gene silencing of Mi PDCD6 mediated by VIGS was revealed. In experimental group, giant cells were plumper, the density of inclusions was higher and the degree of apoptosis was less compared with that of control group, which demonstrated that Mi PDCD6 may promote apoptosis process of giant cells in their host. |
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