Molecular Mechanism Of Helper Component-Proteinase Regulating Sugarcane Mosaic Virus Virulence

Maize dwarf mosaic disease(MDMD)is one of the most serious viral diseases,which has spread to all the maize-growing regions and threatened the safety of maize production in China.Sugarcane mosaic virus(SCMV)is one of the main viral pathogens inducing MDMD in China.SCMV belongs to the genus Potyvirus,and its genome encodes 11 proteins.Potyviral helper component-proteinase(HC-Pro)is the RNA silencing suppressor,and involved in regulating viral virulence.However,the mechanism of HC-Pro regulating SCMV virulence in maize plants is still unclear.In this study,based on the c DNA infectious clone of SCMV(p SCMV),the new residues that regulate virus virulence were identified via reverse genetics.A Zea mays triacylglycerol lipase(ZmTGL)interacted with SCMV HC-Pro was identified via affinity purification coupled with mass spectrometry,and we revealed that ZmTGL inhibited SCMV infection through reducing the accumulation levels and RNA silencing suppression activity of HC-Pro,and activating the salicylic acid pathway.The main findings were read as follow:1.The mutations of arginine(R¹⁸⁴)and lysine(K¹⁸⁶)in FRNK motif of HC-Pro affected SCMV virulence;the spontaneous mutation of glycine at position 440(G⁴⁴⁰)in HC-Pro restored SCMV virulence.The R¹⁸⁴in FRNK motif of SCMV HC-Pro was substituted with isoleucine(I)or K,and the K¹⁸⁶was substituted with alanine(A)or R,respectively,resulting in mutants SCMV-HC_FINK,SCMV-HC_FKNK,SCMV-HC_FRNA,and SCMV-HC_FRNR.Compared with the wild-type SCMV,the accumulation levels of SCMV-HC_FRNRdecreased by about 40%,the accumulation levels of SCMV-HC_FINK,SCMV-HC_FKNK,and SCMV-HC_FRNAdecreased by about 80%in maize plants.At 35 days post-inoculation(dpi),a spontaneous complementary mutation of GGA(codon for G⁴⁴⁰)to AGA(codon for R)in HC-Pro of SCMV-HC_FINKprogeny,resulting in double-mutant SCMV-HC_FINK/G440R.The virulence and HC-Pro RNA silencing suppression activity of SCMV-HC_FINK/G440Rmutant were similar to the wild-type SCMV.The HC-Pro G⁴⁴⁰of SCMV-HC_FINKmutant was substituted with K or histidine(H),resulting in double-mutants SCMV-HC_FINK/G440Kor SCMV-HC_FINK/G440H.The virulence and HC-Pro RNA silencing suppression activity of SCMV-HC_FINK/G440Kmutant were similar to the wild-type SCMV,while the virulence and HC-Pro RNA silencing suppression activity of SCMV-HC_FINK/G440Hmutant were at the similar level as SCMV-HC_FINKmutant.The amino acids corresponding to G⁴⁴⁰of SCMV HC-Pro were asparagine at position 437(N⁴³⁷)for watermelon mosaic virus(WMV)HC-Pro and serine at position 438(S⁴³⁸)for tobacco vein banding mosaic virus(TVBMV)HC-Pro.The substitutions of N⁴³⁷or S⁴³⁸with R could restore HC-Pro RNA silencing suppression activity and the virulence of WMVor TVBMV.2.Mutations were simultaneously introduced into the two residues of HC-Pro FRNK and zinc finger motifs involved in regulating SCMV virulence,and attenuated mutants with genetic stability and effective cross-protection were obtained.The substitutions of cysteine at position 57 or 60(C⁵⁷or C⁶⁰)with A in HC-Pro could decrease its RNA silencing suppression activity and the virulence of SCMV.The SCMV mutants FINK/C57A,FINK/C60A,FKNK/C57A,and FKNK/C60A containing double mutations in HC-Pro were constructed via the substitutions of C⁵⁷or C⁶⁰with A in HC-Pro of SCMV-HC_FINKand SCMV-HC_FKNKmutants.Compared with the wild-type SCMV,the virulence of FINK/C60A,FKNK/C57A,and FKNK/C60A mutants decreased significantly,and caused mild symptoms in maize plants at 60 dpi.FINK/C57A mutant failed to systemically infect maize plants.Cross-protection tests indicated that the cross-protection efficiency of the FKNK/C60A double-mutant reached 100%in maize plants with an interval of 20 days.3.This study revealed that the mechanism of ZmTGL inhibiting SCMV infection through reducing the accumulation levels and RNA silencing suppression activity of HC-Pro,and activating the salicylic acid pathway.To identify potential maize proteins that interacted with SCMV HC-Pro,we inserted a Strep-tag encoding sequence into the 5'-terminus of HC-Pro in p SCMV vector to generate a recombinant SCMV infectious clone p SCMV-Strep-HC-Pro.At 10 dpi,the mosaic symptoms that appeared in the upper leaves of maize plants infected with SCMV-Strep-HC-Pro were similar to those induced by the wild-type SCMV.A ZmTGL protein was identified via affinity purification coupled with mass spectrometry.ZmTGL was clustered into pathogenesis-related lipases(PRLIPs)group.ZmTGL expression level was up-regulated in maize plants infected with SCMV-GFP at 10 dpi.Yeast two-hybrid,luciferase complementation imaging and bimolecular fluorescence complementation assays further confirmed that ZmTGL directly interacted with SCMV HC-Pro in vitro and in vivo.The accumulation levels of SCMV in the ZmTGL-silenced plants were about three times higher than those in the control plants.Over-expression of ZmTGL reduced the RNA silencing suppression activity and the accumulation level of HC-Pro.Mutational analysis found that the reduction of HC-Pro accumulation levels and RNA silencing suppression activity were associated with the lipase hydrolase activity of ZmTGL.Lipase plays an important role in salicylic acid signaling,and over-expression of ZmTGL raised SA accumulation level and up-regulated the expression of SA signaling pathway-related genes in maize plants.The accumulation levels of SCMV in the ZmTGL mutant without lipase hydrolysis activity-expressing maize plants were substantially higher than those in the wild-type ZmTGL-expressing plants,indicating that the lipase hydrolase activity of ZmTGL was critical for inhibiting SCMV infection.In summary,the mutations in HC-Pro FRNK motif affected SCMV virulence,and the spontaneous compensatory mutation G440R in HC-Pro restored virus virulence;SCMV mutants with double-mutations in HC-Pro FRNK and zinc-finger motifs had a reduced probability of reverse mutation.ZmTGL reduced HC-Pro accumulation levels and RNA silencing suppression activity via its lipase hydrolase activity.ZmTGL also activated host SA signaling pathway to inhibit SCMV infection.