| In the rhizosphere,plants interact with countless microorganisms to form various relationships such as symbiosis,parasitism,and competition.Photosynthetic products released by plant roots include soluble sugars,amino acids,organic acids,fatty acids,etc.,which are the main source of soil organic carbon sources and are easily assimilated by microorganisms,attracting microorganisms to colonize plant roots and surrounding areas.There is evidence that plant-derived carbohydrates and fatty acids are utilized by rhizosphere fungi as two distinct forms of carbon sources.However,the respective contributions of these two carbon sources in the symbiotic relationship between fungi and plants,and the mechanism by which fungi utilize plant-derived sugars and fatty acids remain to be further elucidated.In addition to the need to efficiently utilize plant-derived carbon sources for growth,fungi that interact with plants also need to subvert the host immunity.Although the mechanisms by which phytopathogenic fungi evade the plant immune system have been intensively studied,the mechanisms of symbiotic fungi that form symbiotic relationships with plants are poorly understood and needs to be further studied.Metarhizium robertsii has been a good material for studying the interaction of plants,fungi and insects due to its diverse lifestyles(such as saprophytes,plant symbionts,and insect pathogens).At present,the biochemical and genetic mechanisms of M.robertsii infected insects are generally well conducted,but the study on the symbiotic relationship with plants is still in its infancy.In this paper,the mechanism of utilization of plant-derived sugars and fatty acids and the mechanism of evasion of host immunity by M.robertsii were studied,and the main results were listed as follows:1.By analyzing the RNA-Seq data previously published by our lab,we found a monosaccharide transporter MST1 which is highly expressed in saprophytic,parasitic and symbiotic life,and its homologous proteins were found in many plant-associated fungi.Biochemical functional identification indicated that MST1 is an H~+symporter transporter that can transport glucose,fructose,sorbose,mannose,rhamnose and galactose.Compared with the wild-type strain,the conidial germination rate and mycelial growth rate ofΔMst1 in tomato root exudates were reduced,and the ability ofΔMst1 to colonize maize rhizosphere and rhizoplane was also decreased.In addition,MST1 and the oligosaccharide transporter MRT act collectively to colonize the rhizosphere and rhizoplane,and the rhizosphere colonization ability of double-deletion mutantΔMst1::ΔMrt is further reduced compared with single-gene-deletion mutantsΔMst1 orΔMrt.However,the deletion of Mst1 had no significant impact on pathogenicity against insects.2.In addition to the monosaccharide transporter MST1,we also found a protein SUT1 annotated as a sugar transporter on Gen Bank that is specifically expressed in plant roots.There was no significant difference in mycelial growth between the wild-type strain and theΔSut1 in the medium with various monosaccharides as the sole carbon source,but the conidial germination rate and mycelial dry weight of mutantΔSut1 were significantly lower than those of the WT strain in the medium with multiple disaccharides or oligosaccharides(xylose,maltose,sucrose,trehalose,lactose,raffinose)as the sole carbon source.Biochemical functional identification using the monosaccharide transporter mutant of Saccharomyces cerevisiae EBY.VW4000indicated that SUT1 could not transport more than ten kinds of monosaccharides such as glucose,mannose and fructose.Except for glucose,the above-mentioned disaccharides and oligosaccharides failed to elicit electrophysiological responses in heterologous SUT1-expressing Xenopus oocytes in vivo.Compared with the WT strain,the colonization ability ofΔSut1 in Arabidopsis thaliana roots and in the maize rhizosphere and rhizoplane was significantly decreased,indicating that SUT1 plays an important role in the symbiotic relationship between M.robertsii and plants.It was further found that three sugar transporter genes,Mst1,Mrt and Sut1,had a synergistic effect when M.robertsii colonizing maize rhizoplane in soil.3.In addition to sugars,plant-derived fatty acids also play an important role in the symbiotic relationship between M.robertsii and plants.The number of CFU of the WT strain in A.thaliana Col-0 was～1.6-fold higher than A.thaliana fatty acid synthase mutants Kas1 and Kar1(the ability for rhizoplane colonization and endophytic growth is shown as the number of CFU).We further investigated the mechanism of plant fatty acid utilization by M.robertsii.According to the annotation of Gen Bank,there is only one fatty acid transporter in the M.robertsii genome,but the rhizosphere competency of its knock-out mutant is not significantly different from that of the wild-type strain.In the meanwhile,we found that two fatty acid-binding proteins,FABP1 and FABP2,were expressed at high levels in symbiosis with plants.Compared with the wild-type strain,the rhizosphere competency of the knockout mutants ofΔFabp1 andΔFabp2 in wild-type A.thaliana Col-0 had significantly decreased,while their rhizosphere competency in the A.thaliana fatty acid synthesis mutants Kas1 and Kar1 was no significantly difference.Comparing the role of plant-derived sugars and fatty acids in the symbiotic relationship between M.robertsii and plants,it was found that the ability ofΔSut1 to colonize A.thaliana Col-0 was significantly no different from that of the double-gene deletion mutantΔFabp1::ΔFabp2,but the ability ofΔSut1 to colonize A.thaliana fatty acid synthesis mutants was significantly lower than that ofΔFabp1::ΔFabp2.However,the three-gene deletion mutantΔSut1::ΔFabp1::ΔFabp2has a stronger ability to colonize wild-type A.thaliana Col-0 thanΔSut1,and is comparable toΔFabp1::ΔFabp2.It was further found that the double-gene deletion mutantΔFabp1::ΔFabp2 increased the expression levels of the other 6 sugar transporters,and simultaneously knocking out the three genes of Sut1,Fabp1 and Fabp2 further increased the expression of 3 sugar transporters.4.The extracellular protease CSE1 plays an important role in the evasion of host immunity by M.robertsii.The rhizosphere and rhizoplane competency of knock-out mutants of the protease gene Cse1 to maize was significantly reduced compared with the wild-type strain.According to the specific substrate recognition site(Phe-Val-Arg)of CSE1,264 proteins containing CSE1 recognition site were found in the proteins secreted from A.thaliana.GO functional enrichment analysis showed that 22 proteins were localized in plant cell walls(GO:0009505),of which 13 were plant class III cell wall peroxidase(POX).In the symbiotic interactions between A.thaliana andΔCse1,POX activity,ROS content and degree of lignification were higher than those in WT colonized A.thaliana,indicating that CSE1 proteases secreted extracellularly inhibit the initiation of POX-associated defense reactions such as oxidative burst and lignification by inactivating plant cell wall peroxidase.In conclusion,this paper indicated the process of the symbiotic relationship between M.robertsii and plants,and founds that two sugar utilization-related genes and two fatty acid utilization-related genes utilize host-derived carbon sources,and a protease gene that plays an important role in evading host immunity system,revealing a new mechanism for the interaction between M.robertsii and plants. |
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