Molecular Characterization And Functional Analysis Of β-l,4-endoglucanases And Expansins From The Cereal Cyst Nematode Heterodera Avenae

The cereal cyst nematode, Heterodera avenae, is considered as the most economically importantplant-parasitic nematode of cereal crops worldwide, and the average losses owing to H. avenae up to30%。In China, H. avenae is one of most important plant parasitic nematode and distributes throughoutnearly all cereal growing areas. Cloning and identification of parasitism genes is the key tounderstanding the molecular basis on nematode parasitism of plants. Three new β-1,4-endoglucanasegenes (Ha-eng-1a,Ha-eng-2and Ha-eng-3) and one expansin genes (Ha-expb1) expressed in theoesophgeal gland cells of the sedentary cyst nematode, Heterodera avenae, were cloned. The cDNA ofHa-eng-1a is1565bp long (FJ839965) and encodes a deduced463amino acid sequence containing asignal peptide, a catalytic domain and a cellulose binding moudule separated by a linker. The genomicDNA of Ha-eng-1a is1970bp long (JN861118), containing eight introns ranging from56bp to157bp.The cDNA of Ha-eng-2is1215bp long (JN861115) and encodes a deduced333amino acid sequencecontaining a signal peptide and a catalytic domain. The genomic DNA of Ha-eng-2is2460bp long(JN861114), containing seven introns ranging from44bp to610bp. The cDNA of Ha-eng-3is1184bplong (JN861117) and encodes a deduced324amino acid sequence containing a signal peptide and acatalytic domain. The genomic DNA of Ha-eng-3is2673bp long (JN861116), containing seven intronsranging from44bp to610bp. The cDNA of Ha-expb1is1033bp long (HQ386233) and encodes adeduced285amino acid sequence consisit of a signal peptide, a cellulose binding moudule and anexpansin domain. The genomic DNA of Ha-expb1is1700bp long (JN861113), containing five intronsranging from76bp to310bp. Southern blot analysis revealed that both Ha-eng-3and Ha-expb1could bea member of a small multigene family. In situ hybridization showed that the four cloned parasitismgenes all accumulated specifically in the two subventral gland cells of second stage juveniles of H.avenae. Semiquantitive RT-PCR analysis confirmed that their transcriptions were strong at thepre-parasitic and early parasitic second-stage juveniles, and low at the late parasitic stages of thenematodes. Recombinant proteins HA-ENG-1A, HA-ENG-2, HA-ENG-3and HA-EXPB1wereobtained by heterologus expression and detected by SDS-PAGE.The cellulase activity of recombinantβ-1,4-endoglucanase proteins HA-ENG-1A, HA-ENG-2and HA-ENG-3were evidenced in vitro,indicationg these proteins are functional. Knocking down Ha-eng-2using RNA interference resulted ina significant reduction of nematode infecitivity by40%. These lines of results indicatied that theisolated β-1,4-endoglucanases and expansin could be secreted into plant tissues by H. avenae and play acrucial role in plant cell wall degradation during penetration and migration of neamtodes in the hostplants.