Purification, Gene Cloning And Function Characterization Of A Novel Elicitor―MoHripl From Magnaporthe Oryzae

Elicitors are molecules which play a key role in the induction of plant defense responses andenhanced plant disease resistance against its pathogens. In this article, a protein elicitor wasisolated and purified from the culture filtrate of rice blast pathogenic fungus—Magnaportheoryzae. This elicitor could induce a hypersensitive response and early events in tobacco cell. Thenthe gene of this protein elicitor was cloned and expressed in Escherichia coli. Recombinantprotein could also induce a hypersensitive response in tobacco. The molecular mechanism ofresistance induced by elicitor in rice was study by application of real-time PCR. And the datademonstrated that the SA signaling pathway was the mainly pathway in the induction of rice plantdefense response. This study revealed that the elicitor could induce resistance in rice and provideuseful information for further study in mechanisms of this protein elicitor. The results of thisarticle are below:1. The protein elicitor from the culture filtrate of rice blast pathogenic fungus--Magnaportheoryzae, was isolated by ammonium sulfate precipitation, AKTA Protein Purification System,native-PAGE and electroelution. The pure protein showed a single band on SDS-PAGE gel and asingle point on2D-PAGE with relative molecular mass14kD. We designated this proteinMagnaporthe oryzae hypersensitive response-inducing protein1（MoHrip1）.2. MoHrip1elicitor could induce a hypersensitive response in tobacco leaves, with aminimum concentration of5μM. MoHrip1was shown to be a pure protein with no carbohydratemoieties. pI=4.53.3. We got three pieces of amino acid sequences from MoHrip1by using de novo sequencing.Similarity matching by protein BLAST search indicated that the protein presented high identity toa conserved hypothetical protein （GenBank accession number: XP₃66602） from the M. oryzaestrain70-15. The hypothetical protein contains142amino acids with a theoretical molecularweight of14.322kD.4. We cloned the gene of MoHrip1and got the pure recombinant protein, which could alsoinduce the HR in tobacco leaves.5. MoHrip1elicitor could trigger defense responses in tobacco plant and induce early events（ROS burst, NO accumulation, callose deposition, phenolics metabolism and Extracellularmedium alkalization）. In other way, it demonstrates that MoHrip1is a real protein elicitor.6. MoHrip1elicitor could induce resistance against rice blast in rice seedlings. After3daysof treatment with MoHrip1elicitor, rice seedlings could be obviously resistant against infection ofM. oryzae. Expression of pathogenesis-related genes （OsPR-1a and OsPR-10a） was induced byMoHrip1, suggesting that may be a mechanism of induced plant resistance.7. Detection of the expression of key genes in defense signaling pathway by real-time PCRdemonstrated that SA signaling pathway is a main pathway involving in the defense responseinduced by MoHrip1elicitor.