Colonization Process Of Cotton Leaves And Monocotyledon By GFP-Tagged Verticillium Dahliae

Cotton is widely cultivated because of its economically valuable fibers and oil seeds. Over 30% area of upland cotton are subject to Verticillium wilt caused by Verticillium dahliae (V. dahliae), and the disease has become the most economically important disease of upland cotton. In general, the pathogenic fungi infect the root of the plant directly in the soil and enter the vessel through the cortical cells, in which the spores and mycelia of pathogens block the vessel of the plant. In addition, V. dahliae toxins and acidic glycoproteins produced by the pathogens, are also important pathogenic factors that can make the plant wilt quickly. Reports on foliage infection by soilborne pathogens are not common. In this study, we observed the infection and colonization process of V. dahliae on cotton leaves, activation of defence responses and the infection on graminaceous monocots rice and maize.We observed the infection process of nondefoliating V1070-GFP on susceptible cotton cultivar Sumian 22 leaves. Germination of nondefoliating V1070-GFP conidia on the isolated abaxial epidermis of susceptible cotton cultivar Sumian 22 started at 2 hours post inoculation(hpi). Then the hyphae prolonged and penetrated into epidermal cells without forming any infection structure, several hyphae penetrated through stomata. Morover, hyphae within epidermal cells penetrated back into air directly or through stomata. To observe the spreading of V1070-GFP conidia on alive leaves, a 10 ul droplet of the conidial suspension was spotted on the cotyledons surface on either side of the leaf margin. Following applications of the GFP-expressing transformant of V1070-GFP on leaves of Sumian 22, green fluorescence was observed 24 hours after treatment. By 48 hpi, necrosis appeared in infected sites and enlarged with time flow. Conidia on the infection sites began to germinate and continued elongating from the necrotic part. The the hyphae continued spreading downwards from the surface of cotton leave, and arrived stems and roots. On cotton stem, the amount of hyphae on the hypocotyl was higher than the rest parts of the stem. In addition, large numbers of macroconidia and microsclerotia produced on the surface of hypocotyl. In the following days, the macroconidia and microsclerotia regerminated and penetrated transversely into epidermis, cortex and arrived into xylem vessel of hypocotyl. We also isolated V1070-GFP from the infected leaves, and found that the quantity of V1070-GFP increased quikly at the first 10 days. During 10 to 30 days, the quantity of V1070-GFP increased slowly and kept stable.Using aniline blue staining and DAB staining method to observe the accumulation of callose and hydrogen peroxide in cotton leaves after inoculated with V. dahliae. We found that callose deposited at the infection site and the amount of callose increased gradually. After 24 hpi, hydrogen peroxide increased significantly in infected leaves. Then we detected the gene expression of PR1, LOX and WRKY4 genes by real-time PCR. The results showed that gene expression of all three genes increased with different degrees when induced by pathogen V1070-GFP.Onion epidermis was used to observe germination and infection process of V. dahliae V1070-GFP conidia. The conidia of V1070-GFP can geminate and expand along the junctions of onion epidermal cells. However, the germinated and expanded rate was slower than on cotton epidermis, and hyphae could not penetrate into the epidermal cells. Then we observed the process of V. dahliae on rice and maize roots by stereo microscope, and isolated V1070-GFP from the rice and maize roots. The adsorption of V. dahliae on rice and maize roots was not observed, and none of V1070-GFP was seperated from them. Besides, we also observed the infection process of V. dahliae on rice and maize leaves. Most of the V1070-GFP spore suspension covered on the surface of rice and maize leaves, and could not be absorbed. Conidia would not germinate from these parts. Few of spore suspension were absorbed, and the conidia germinated from here. However, the hyphae can not penetrate into the mesophyll cells and cause lesions on rice and maize leaves.sGFP encoding green fluorescent protein was translated into defoliating V. dahliae isolate VD07038 and mCherryRFP encoding red fluorescent protein was translated into non-defoliating V. dahliae Bp2 via Agrobacterium tumefaciens-mediated transformation. Finally, positive transformants tagged by green fluorescent protein or red fluorescent protein were obtained. The hygromycin B genetic stability of transgenic progenies was tested by series culture and molecular verification. According to colony morphology, growth rate and pathogenicity, we found that traits of most of transformants were similar to those of wild-type, few transformants tended to form mutants. Using the GFP-tagged V. dahliae VD07038G-10 isolate, colonization of the fungus in roots of the susceptible cotton sumian22 was investigated. As the results showed that, twelve hours after inoculation via the root dipping methods, roots surface of Sumian 22 were covered with conidia,7-9 days after inoculation, hyphae from the leading edge of the colony progressed acropetally up the xylem vessels of infected roots.This study demonstrated that V. dahliae can infect leaves of cotton seedlings, and then systermatically colonized stems and roots, resulting in the plant wilt; After V. dahliae infection, the cotton leaves would produce a series of defence reaction against its infection; V. dahliae can germinate on the leaves of maize and rice, but it can not penetrate into the inner tissue of leaves, Indicating that a recognition mechanism exist between V. dahliae and its host. These results will provide theoretical basis for V. dahliae cycle and pathogenic mechanism.