The Anti-TMV Activities Triggered By Trichokonins, And The Mechanisms Of Arabidopsis Root Development Involved In Trichokonin Ⅵ

Trichoderma species belong to saprophytic fungi in Deuteromyeotina. They are common in root, soil and foliar environments. It has been known for many years that they can control fungal and bacterial phytopathogens. Plant virus diseases are a kind of important diseases in agricultural production, and usually cause heavy losses. The use of chemical control agents will lead to plant viral drug-resistance, and thus significantly reduce the effects of these control agents. Microbe is a natural source providing many kinds of various secondary metabolites with antiviral activity, which provides a new way for control of plant virus diseases. Biocontrol agents T. harzianum T-1 and T. virens T3 are found to have anti-virus activity against green mosaic virus when they colonized cucumber root. Although the antimicrobial activity of Trichoderma spp. against fungal or bacterial phytopathogens and the involved mechanisms have been widely studied, the antiviral effect of Trichoderma spp. and the exact mechanisms underlying the process are still largely unknown. Peptaibols are a class of antibiotic peptides mainly synthesized by Trichoderma. Many peptaibols are shown to have a broad spectrum of antimicrobial activity against variety of fungal and bacterial phytopathogens.In our previous work, a kind of antimicrobial peptaibols named as Trichokonins (TKs) were isolated and purified from T. pseudokoningii SMF2. These TKs have been demonstrated to exhibit antimicrobial activity against a range of Gram-positive bacterial and fungal phytopathogens. However, the antiviral activity of TKs and their mechanisms involved in plant resistance pathways are still unknown. Only Peptaivirins A and B from Apiocrea spp. have been reported to have inhibitory activity against tobacco mosaic virus (TMV) infection to tobacco. In order to investigate the anti-virus activity of TKs and the mechanisms underlying the process, we chose TMV-resistant (NN) plants of tobacco (Nicotiana tabacum L. cv. Samsun) as the study object to study the anti-TMV activity of TKs.Besides the well-known biocontrol activity, the enhancement of plant growth by Trichoderma has been known for many years. Until recently, it is becoming increasingly clear that many Trichoderma strains also have substantial direct influence on plant development and crop productivity. However the effects of peptaibols on plant growth are largely unknown.In order to learn the effect of peptaibols on plant growth and development, we took TK VI, the main component of TKs produced by T. pseudokoningii SMF2, as the representative of peptaibols to study the effect of TK VI on plant growth and development. Our work emphasized on the promotive effect of TK VI on Arabidopsis root growth. Furthermore, we deeply analysed its involved mechanisms.This paper studied the anti-TMV activities triggered by TKs, and the mechanisms of Arabidopsis root development involved in TK IV. The main results are as follows:Ⅰ. Study on the control of tobacco mosaic virus by TKs1. Deactivation of TMV particles by TKs. The inactivation activity of the typical plant virus TMV by TKs was detected in vitro. The inhibitory activity of TKs against TMV was estimated by half-leaf method and the untreated TMV virons was used as the control. The inhibitory rate analysis revealed that the inhibitory rate of TMV increased to 23.23% with 100 nM TKs treatment in contrast with the control. When the dose of TKs was enhanced to 20μM, the inhibitory rate increased to 58%. We also observed the changes in the TMV particles caused by TKs treatment using transmission electron microscope. TMV particles became ruptured and abnormal after TKs treatment. These results showed that TKs could deactivate TMV particles in vitro and the inactivation ability was dose-dependent.2. Defense resistance in tobacco induced by TKs and the involved mechanisms.Tabacco plants were sprayed with increasing concentrations of TKs. After 4 d induction, plants were inoculated with TMV. The lesion inhibition ratio in tobacco treated with 50 nM,100 nM and 200 nM TKs was 15%,54%and 35%, respectively, revealing that tobacco resistance against TMV was significantly improved after TKs treatment. After the same challenged time, tobacco plants treated with 100 nM TKs showed obviously higher lesion inhibition ratio, smaller average final lesion diameter and smaller lesion area than the control, indicating that TKs mounted resistance in tobacco against TMV infection.TKs treatment increased production of reactive oxygen species and formation of phenolic compounds in tobacco, which indicated that TKs-induced resistance against TMV involved a timely induction of early plant defense reactions. TKs treatment also led to significant increase in the activities of PAL, POD and PPO, suggesting that TKs induced PAL, POD and PPO involved defense responses in tobacco against TMV. Moreover, Up-regulation of the expression of selected plant defense genes in tobacco suggested that TKs-induced resistance required cross talk among defense pathways. Taken together, the results indicated that TKs are involved in plant resistance against TMV through virus inactivation and activation of multiple plant defense pathways. This represents the first report about the involved mechanism of peptaibols against plant virus, which may imply the potential of peptaibols in plant viral disease control.Ⅱ. Study on the effect of TK VI on root development and its involved mechanisms.1. Effect of TK VI on the growth and development of plants.We tested the effect of TK VI on the growth of Arabidopsis of different ecotypes (Col, Ws and Ler) by growing plants on 1/2 Murashige and Skoog solid medium containing different concentrations of TK VI. After 14 d of growth, treatments of 50 nM TK stimulated shoot and root biomass production of all ecotype seedlings. Seedlings of Col ecotype showed the most sensitve response to TK VI. We observed that TK VI treatment had a dramatic effect on the Arabidopsis root system architecture by altering primary root growth and emerged lateral root number. When the concentration of TK VI was increased to 5μM, TK VI had an inhibitory effect on root growth. To further investigate the effects of TK VI on plant development, primary root length, number of emerged lateral roots, and lateral root density were determined in Arabidopsis seedlings treated with various concentrations of TK VI. After 9 d of growth, primary root growth promotion was observed, indicating the growth rate maintained stable during the promoting process. Increase of number of lateral roots caused by TK VI could be due to a stimulation of the emergence of pre-existing lateral root primordial or to the de novo formation of additional root primordia. To more closely analyze the effects of TK VI on lateral root development, lateral root primordia (LRP) originatd from the Arabidopsis primary root were quantified. The lateral root density and the LRP number analysis revealed that TK VI had no appreciable impact on plant LRP. These results suggested that the growth promotion effect of TK IV is independent of auxin synthesis.We also studied the effect of TK VI on tobacco, alfalfa, maize, rice and wheat. Similar to Arabidopsis, treatments with a low concentration TK VI (5-50 nM) promoted shoot and root biomass production of tobacco and alfalfa. But different from those dicotyledons, TK VI had an inhibitory effect on the growth of maize, rice and wheat. Those results indicated that TK VI has higher phytotoxicity to monocotyledons than to dicotyledons at same concentrations.2. The involved mechanism of TK VI induced growth promotion of ArabidopsisTo test whether TK VI treatment could alter root development by altering cell division and/or elongation, we analyzed the expression of the cell cycle marker CycBl::GUS in transgenic plants with TK VI treatment. The expression of GUS in the seedlings with 50 nM TK VI treatment was higher than that observed in the untreated seedlings, which indicated an enhanced cell division. To analyze whether cell elongation was triggered by TKⅥ, epidermal cell size in the maturation region of primary roots was measured. These measurements showed that epidermal cells in plants treated with 50 nM TKⅥwere 2 folds longer than those in the untreated plants.The phytohormone auxin is a key regulator of root development. To determine if TKⅥinvolved in an auxin-related signaling pathway, we conducted to analyze the expression of the auxin-inducible DR5::GFP gene marker. The GFP expression pattern in DR5::GFP seedlings treated with 50 nM TKⅥexhibited diffuse distribution in primary root tip, which was similar to the pattern of the polar auxin transport inhibitor NPA (N-1-naphthylphthalamidic acid). Therefore, we concluded that TKⅥtreatment can alter the polar transportion of auxin. To evaluate the effects of TKⅥon auxin-signaling pathway, mutants were used to investigate the expression pattern of auxin transporters. Compared with Col-0 seedlings, root elongation was also observed on the auxin-efflux mutant eir1-1. This result suggested that TKⅥmight have effect on auxin-efflux carrier. We further detected whether basipetal/acropetal auxin flow was involved in this process. By using PINs::GFP gene markers, we found the expression of PIN1 was increased and the expression of PIN2 was decreased. Finally, our studies showed that TKⅥinduced root growth promotion through abnormal auxin polar transport involved in PINs in Arabidopsis.