A novel partitivirus conferring hypovirulence by affecting vesicle transport in the fungus Colletotrichum.

Colletotrichum spp. are economically important phytopathogenic fungi that cause anthracnose in a variety of plant species worldwide. Hypovirulence-associated mycoviruses provide new options for the biological control of plant fungal diseases. Here, we found a novel partitivirus from Colletotrichum alienum and named it Colletotrichum alienum partitivirus 1 (CaPV1). CaPV1 contained two dsRNA segments encoding an RNA-dependent RNA polymerase and a capsid protein and was classified under the genus Gammapartitivirus of the family Partitiviridae. CaPV1 significantly decreased host virulence, mycelial growth, appressorial development, and appressorium turgor but increased conidial production with abnormal morphology. In addition, CaPV1 could be successfully transfected into other Colletotrichum species, including C. fructicola, C. spaethianum, and C. gloeosporioides, and caused hypovirulence, indicating the broad application potential of this virus. CaPV1 caused significant transcriptional rewiring of the host fungus C. alienum. Notably, some genes related to vesicle transport in the CaPV1-infected strain were downregulated, consistent with the impaired endocytosis pathway in this fungus. When the Rab gene CaRab7, which is associated with endocytosis in vesicle transport, was knocked out, the virulence of the mutants was reduced. Overall, our findings demonstrated that CaPV1 has the potential to control anthracnose caused by Colletotrichum, and the mechanism by which Colletotrichum induces hypovirulence is caused by affecting vesicle transport.IMPORTANCEColletotrichum is a kind of economically important phytopathogenic fungi that cause anthracnose disease in a variety of plant species worldwide. We found a novel mycovirus of the Gammapartitivirus genus and Partitiviridae family from the phytopathogenic fungus Colletotrichum alienum and named it CaPV1. This study revealed that CaPV1 infection significantly decreased host virulence and fitness by affecting mycelial growth, appressorial development, and appressorium turgor. In addition, CaPV1 could also infect other Colletotrichum species, including C. fructicola, C. spaethianum, and C. gloeosporioides, by viral particle transfection and resulting in hypovirulence of these Colletotrichum species. Transcriptomic analysis showed that CaPV1 caused significant transcriptional rewiring of the host fungus C. alienum, especially the genes involved in vesicle transport. Moreover, endocytosis and gene knockout assays demonstrated that the mechanism underlying CaPV1-induced hypovirulence is, at least in part, caused by affecting the vesicle transport of the host fungus. This study provided insights into the mechanisms underlying the pathogenesis of Colletotrichum species and mycovirus-fungus interactions, linking the role of mycovirus and fungus vesicle transport systems in shaping fungal pathogenicity.

Novel and diverse mycoviruses co-infecting a single strain of the phytopathogenic fungus Alternaria dianthicola.

Alternaria dianthicola is a pathogenic fungus that causes serious leaf or flower blight on some medicinal plants worldwide. In this study, multiple dsRNA bands in the range of 1.2-10 kbp were found in a Alternaria dianthus strain HNSZ-1, and eleven full-length cDNA sequences of these dsRNA were obtained by high-throughput sequencing, RT-PCR detection and conventional Sanger sequencing. Homology search and phylogenetic analyses indicated that the strain HNSZ-1 was infected by at least nine mycoviruses. Among the nine, five viruses were confirmed to represent novel viruses in the families Hypoviridae, Totiviridae, Mymonaviridae and a provisional family Ambiguiviridae. Virus elimination and horizontal transmission indicated that the (-) ssRNA virus, AdNSRV1, might be associated with the slow growth and irregular colony phenotype of the host fungus. As far as we know, this is the first report for virome characterization of A. dianthus, which might provide important insights for screening of mycovirus for biological control and for studying of the interactions between viruses or viruses and their host.

Characterization of a Novel Ourmia-Like Mycovirus Infecting Magnaporthe oryzae and Implications for Viral Diversity and Evolution.

Here, the molecular characterization of a novel mycovirus that was isolated from a phytopathogenic fungus Magnaporthe oryzae and designed as Magnaporthe oryzae ourmia-like virus 4 (MOLV4) is reported. MOLV4 has a genome that is 2497 bp long and possesses a single open reading frame (ORF), which encodes the product RNA-dependent RNA polymerase (RdRp). Sequence similarities were found between the MOLV4 encoded RdRp and the counterparts of a few previously reported ourmia-like mycoviruses. Virus-curing and biological comparison indicate that the virus has no or mild effects on the morphology and mycelium growth rate of the host fungus. Phylogenetic analysis using the RdRp aa sequences was performed. The results show that MOLV4 is clustered with the ourmia-like mycoviruses, forming a clade closely related to ourmiaviruses but distinct from narnaviruses. In addition, database searches revealed that several MOLV4-related sequences are present in the transcriptome shotgun assembly (TSA) library, expressed sequence tag database (ESTdb), whole-genome shotgun (WGS) library, and genomic survey sequences (GSS) libraries of a few other species of eukaryote organisms. Our results show that MOLV4, together with other similar ourmia-like mycoviruses, might represent a virus clade that links the plant ourmiaviruses and fungal narnaviruses and has a wide range of hosts.

A Victorivirus and Two Novel Mitoviruses Co-Infected the Plant Pathogen Nigrospora oryzae.

Three dsRNAs, in sizes of approximately 2.5⁻5 kbp, were detected in the plant pathogenic fungus Nigrospora oryzae strain CS-7.5-4. Genomic analysis showed that the 5.0 kb dsRNA was a victorivirus named as Nigrospora oryzae victorivirus 2 (NoRV2). The genome of NoRV2 was 5166 bp in length containing two overlapping open reading frames (ORFs), ORF1 and ORF2. ORF1 was deduced to encode a coat protein (CP) showing homology to the CPs of viruses belonging to the Totiviridae family. The stop codon of ORF1 and the start codon of ORF2 were overlapped by the tetranucleotide sequence AUGA. ORF2 was predicted to encode an RNA-dependent RNA polymerase (RdRp), which was highly similar to the RdRps of victoriviruses. Virus-like particle examination demonstrated that the genome of NoRV2 was solely encapsidated by viral particles with a diameter of approximately 35 nm. The other two dsRNAs that were less than 3.0 kb were predicted to be the genomes of two mitoviruses, named as Nigrospora oryzae mitovirus 1 (NoMV1) and Nigrospora oryzae mitovirus 2 (NoMV2). Both NoMV1 and NoMV2 were A-U rich and with lengths of 2865 and 2507 bp, respectively. Mitochondrial codon usage inferred that each of the two mitoviruses contains a major large ORF encoding a mitoviral RdRp. Horizontal transfer experiments showed that the NoMV1 and NoMV2 could be cotransmitted horizontally via hyphal contact to other virus-free N. oryzae strains and causes phenotypic change to the recipient, such as an increase in growth rate. This is the first report of mitoviruses in N. oryzae.

Diverse, Novel Mycoviruses From the Virome of a Hypovirulent Sclerotium rolfsii Strain.

Sclerotium rolfsii, which causes southern blight in a wide variety of crops, is a devastating plant pathogen worldwide. Mycoviruses that induce hypovirulence in phytopathogenic fungi are potential biological control resources against fungal plant diseases. However, in S. rolfsii, mycoviruses are rarely reported. In a previous study, we found a hypovirulent strain carrying a diverse pattern of dsRNAs. Here, we utilized the RNA_Seq technique to detect viral sequences. Deep sequencing, RT-PCR and Sanger sequencing validation analyses revealed that this strain harbors various new viral species that show affinity to the distinctly established and proposed families Benyviridae, Endornaviridae, Fusariviridae, Hypoviridae, and Fusagraviridae. Moreover, some viral sequences that could not be assigned to any of the existing families or orders were also identified and showed similarities to the Alphavirus, Ourmiavirus, phlegivirus-like and Curvularia thermal tolerance virus-like groups. In addition, we also conducted deep sequencing analysis of small RNAs in the virus-infecting fugal strain. The results indicated that the Dicer-mediated gene silencing mechanism was present in S. rolfsii. This is the first report of viral diversity in a single S. rolfsii fungal strain, and the results presented herein might provide insight into the taxonomy and evolution of mycoviruses and be useful for the exploration of mycoviruses as biocontrol agents.

Mycoviruses in the Plant Pathogen Ustilaginoidea virens Are Not Correlated with the Genetic Backgrounds of Its Hosts.

Ustilaginoidea virens, the causal agent of rice false smut, is one of the most devastating grain diseases that causes loss of yield in most rice-growing areas worldwide. In this study, we performed a dsRNA screen to isolate mycoviruses from 35 U. virens strains. The results revealed that 34 of the tested isolates were infected by various dsRNA elements, displaying highly viral diversity and mixed infections. We characterized a 5.3 kbp dsRNA from a typical isolate containing dsRNA segments with sizes ranging from 0.5 to 5.3 kbp. Sequence analysis of its genomic properties indicated that it is a novel victorivirus, named Ustilaginoidea virens RNA virus 5 (UvRV5), that belongs to the family Totiviridae. RT-PCR detection was performed and indicated that not all the dsRNA bands that were 5.3 kbp in size contained UvRV5. Moreover, the genetic relatedness of all the U. virens strains was estimated according to phylogenetic analysis of the partial intergenic spacer region (IGS) sequences. However, concordance was not found between the dsRNA profiles and the IGS-based genetic relatedness of their host fungi.

Molecular Characterization of a Trisegmented Mycovirus from the Plant Pathogenic Fungus Colletotrichum gloeosporioides.

A novel double-stranded RNA (dsRNA) mycovirus, consisting of three dsRNA genome segments and possibly belonging to the family Chrysoviridae, was isolated from the filamentous phytopathogenic fungus Colletotrichum gloeosporioides and designated as Colletotrichum gloeosprioides chrysovirus 1 (CgCV1). The three dsRNAs of the CgCV1 genome with lengths of 3397, 2869, and 2630 bp (dsRNAs1-3) were found to contain a single open reading frame (ORF) putatively encoding the RNA-dependent RNA polymerase (RdRp), a capsid protein, and a protease, respectively, all of which exhibited some degree of sequence similarity to the comparable putative proteins encoded by the genus Chrysovirus. The 5'- and 3'-untranslated regions in each dsRNA segment contained similar sequences that were strictly conserved at the termini. Moreover, isometric virus-like particles (VLPs) with a diameter of approximately 40 nm were extracted from fungal mycelia. Phylogenetic analysis based on the conserved dsRNA1-encoded RdRp showed that CgCV1 is a new virus belonging to the Chrysoviridae family. BLAST analysis revealed the presence of CgCV1-like sequences in the chromosomes of Medicago truncatula and Solanum tuberosum. Moreover, some sequences in the transcriptome shotgun assembly (TSA) library and expressed sequence tag database (ESTdb) of other eudicot and monocot plants were also found to be related to CgCV1.

A novel single-stranded RNA virus isolated from the rice-pathogenic fungus Magnaporthe oryzae with similarity to members of the family Tombusviridae.

Here, we report a novel virus isolated from rice blast fungus, Magnaporthe oryzae, an important plant pathogen. This virus has an RNA genome of 3246 nucleotides. Its genome possesses two in-frame open reading frames (ORFs). The smaller ORF1 encodes a protein with significant similarity to a protein encoded by the ssRNA mycovirus Diaporthe ambigua RNA virus 1 (DaRV1). The larger ORF2 encodes a protein with similarity to RNA-dependent RNA polymerases (RdRp) of DaRV1 and other plant viruses of the family Tombusviridae. In silico analysis and comparisons with DaRV1 genome expression suggest that ORF2 is translated via a readthrough mechanism together with ORF1. Based upon results of this study, this virus, for which the provisional name Magnaporthe oryzae virus A (MoVA) is proposed, belongs to a new virus species. Furthermore, MoVA along with DaRV1 belong to a new taxon of mycoviruses that are evolutionarily related to plant viruses belonging to the family Tombusviridae.

Characterization of a novel single-stranded RNA virus, closely related to fusariviruses, infecting the plant pathogenic fungus Alternaria brassicicola.

The alternaria blackspot of rapeseed is one of the most prominent diseases of rapeseed. It is caused by three species of the genus Alternaria: Alternaria brassicicola, Alternaria brassicae, and Alternaria raphanin. Here we report a novel positive-sense RNA virus from an A. brassicicola strain 817-14. The virus has a 6639 nucleotide (nt) long genome, excluding a poly (A)-tail, and was predicted to contain three putative open reading frames (ORF1, ORF2, and ORF3). The large ORF1 encoded a 174-kDa polyprotein (composed of 1522 amino acid residues) containing a conserved RNA-dependent RNA polymerase (RdRp) domain and a helicase domain. The other two smaller ORFs encoded polypeptides with unknown function. Homology search and phylogenetic analysis, based on the RdRp and helicase domains, suggest that this virus is related to and grouped with Sclerotinia sclerotiorum fusarivirus 1 (SsFV1), Rosellinia necatrix fusarivirus 1 (RnFV1), Fusarium graminearum virus-DK21 (FgV1), and Penicillium roqueforti RNA mycovirus 1 (PrRV1), all of which belong to a newly proposed family Fusariviridae. For this study, we designed the virus as "Alternaria brassicicola fusarivirus 1" (AbFV1). Virus elimination revealed that AbFV1 has no conspicuous impact on the biological properties of its host.

Genome sequence of a novel mycovirus of Rhizoctonia solani, a plant pathogenic fungus.

Here we present the genome sequence of a novel dsRNA virus we designed as Rhizoctonia solani RNA virus HN008 (RsRV-HN008) from a filamentous fungus R. solani. Its genome (7596 nucleotides) contains two non-overlapping open reading frames (ORF1 and ORF2). ORF1 encoded a 128 kDa protein that showed no significant identity to any other virus sequence in the NCBI database. ORF2 encoded a protein with a molecular weight of 140 kDa and shared a low percentage of sequence identity to the RdRps of unclassified dsRNA viruses. Sequence analysis revealed that RsRV-HN008 may be a member of a novel unclassified family of mycoviruses.

A novel mycovirus identified from the rice false smut fungus Ustilaginoidea virens.

The complete sequence of a novel mycovirus infecting Ustilaginoidea virens, the causal agent of false smut of rice, is reported here and designated as Ustilaginoidea virens unassigned RNA virus HNND-1 (UvURV-HNND-1). This virus has an undivided dsRNA genome of 2903 nt in length and contains two non-overlapping open reading frames (ORF1 and 2), with the small ORF1 encoding a protein of unknown function that showed sequence similarity to the comparable protein in virus Alternaria longipes dsRNA virus 1(AlRV1) and a larger ORF2 encoded the protein showing identities to the RNA-dependent RNA polymerases of AlRV1 and some other unassigned dsRNA viruses. Phylogenetic analysis showed that UvURV-HNND-1 is more closely related to unclassified viruses such as AlRV1 and distinct from distantly related members of the family Partitiviridae. Here, we propose in accordance with previous reports that UvURV-HNND-1 might belong to a new mycovirus genus together with AlRV1 and other similar viruses.

Genome sequence of a novel endornavirus from the phytopathogenic fungus Alternaria brassicicola.

In an effort to discover new mycoviruses from phytopathogenic fungi, a dsRNA molecule of 10,290 nt, resembling those associated with the viruses belonging to the family Endornaviridae, was isolated from Alternaria brassicicola, one of the causal agents of rapeseed black spot disease. Genome analysis revealed the presence of a single open reading frame coding for a polyprotein of 3400 aa containing conserved viral methyltransferase (MTR), viral RNA helicase 1 (Hel-1), and RNA-dependent RNA polymerase (RdRp) domains. In addition, a cysteine-rich region (CRR) with conserved CXCC motifs, shared among several endornaviruses, was also identified between the MTR and Hel-1 domains. Phylogenetic analysis based on the RdRp sequence strongly suggested that the virus infecting A. brassicicola should be considered a representative of a novel endornavirus species, and this virus was designated as Alternaria brassicicola endornavirus 1 (AbEV1).

The complete nucleotide sequence and genomic organization of a novel victorivirus with two non-overlapping ORFs, identified in the plant-pathogenic fungus Phomopsis vexans.

In this study, a novel virus designated Phomopsis vexans RNA virus 1 (PvRV1) was identified in a strain of Phomopsis vexans. The complete genomic nucleotide sequence was determined and analyzed. Sequence analysis indicated that PvRV1 is closely related to viruses in the genus Victorivirus of the family Totiviridae. Two open reading frames (ORF1 and 2) were found in the PvRV1 sequence, and these showed significant similarity to the capsid protein (CP) and RNA-dependent RNA polymerase (RdRp), respectively, of members of the family Totiviridae. The two ORFs were spaced 98 nt apart, which is unique to PvRV1 and different from the overlapping arrangement in most victoriviruses. The expression strategies of the CP and RdRp are discussed based on in silico RNA secondary structure analysis.

Detection and sequence analysis of two novel co-infecting double-strand RNA mycoviruses in Ustilaginoidea virens.

Four novel double-stranded RNA molecules, named dsRNA 1 (5124 bp), dsRNA 2(1711 bp), dsRNA 3 (1423 bp) and dsRNA 4 (855 bp), were detected in strain HNHS-1 of Ustilaginoidea virens, the causal agent of rice false smut disease. Sequence analysis showed that the dsRNA1 contains two overlapping open reading frames (ORF) potentially encoding proteins with modest levels of sequence similarity to the coat protein (CP) and putative RNA-dependent RNA polymerase (RdRp), respectively, of viruses of the family Totiviridae. The deduced gene product of the ORF encoded by dsRNA2 is homologous to putative RdRp of viruses in the family Partitiviridae; the ORF encoded by dsRNA3 shares some similarity to a hypothetical protein with unknown function. It is noteworthy that the dsRNA4 lacked integrated ORFs. Isomeric viral particles of about 40 nm in diameter were observed by transmission electron microscopy in a mycelium tissue preparation of strain HNHS-1-R1, a single-spore subculture of strain HNHS-1 containing only the dsRNA1 segment. Phylogenetic analysis and examination of the organization of the two putative RdRp sequences both indicated that there are at least two novel virus species present in strain HNHS-1. We named the two novel viruses Ustilaginoidea virens RNA virus 2 and Ustilaginoidea virens partitivirus 4, respectively.

Complete genome sequence and organization of a novel virus from the rice false smut fungus Ustilaginoidea virens.

In this study, three dsRNA segments from the rice false smut fungus Ustilaginoidea virens, the causal agent of a serious disease in rice, with molecular size ranging from 1.3 to 5 Kb, were isolated and named as dsRNA-L, dsRNA-M, and dsRNA-S. The complete nucleotide sequences of dsRNA-M and dsRNA-S were determined and analyzed. The dsRNA-M putatively encodes an RNA-dependent RNA polymerase, which is similar to that of the partitiviruses in the family Partitiviridae. Although the protein encoded by dsRNA-S showed less similarity to the typical coat protein of the virus in the family Partitiviridae, the structural analysis results indicated that the dsRNA-S might function as the capsid protein. We propose that the virus is Ustilaginoidea virens partitivirus 2-Uv0901, a new member, but distantly related to the newly proposed genus Gammapartitivirus with a distinct sequence pattern of capsid protein.

[Analysis of differential protein expression of REMI mutant of Trichoderma koningii].

On purpose of expounding the phenotype change of Trichoderma mutant which produces cellulase, protein profiles of T. koningii CICC and its mutant TK-2R-1 were detected by 2-DE. Eight unique spots were found and four of them were identified and their functions by MS-TOF-TOF were connected with growth and metabolization of Trichoderma spp. The research provides a new theoretical basis and methods for the construction of Trichoderma mutant which is used to produce efficient cellulase.

Transcription factor WRKY70 displays important but no indispensable roles in jasmonate and salicylic acid signaling.

The transcription factor WRKY70 was previously reported to be a common component in salicylic acid (SA) and jasmonate (JA) mediated signal pathways in Arabidopsis. Here, we present that the inactivation of the WRKY70 gene in wrky70-1 mutant does not alter the responses of both JA and SA, and that wrky70 mutation is unable to restore the coi1 mutant in JA responses. However, overexpression of WRKY70 reduces JA responses such as expression of JA-induced genes and JA-inhibitory root growth, and activates expression of SA-inducible PR1. These data indicate that the WRKY70 is important but not indispensable for JA and SA signaling, and that other regulators may display the redundant role with WRKY70 in modulation of JA and SA responses in Arabidopsis. Furthermore, we showed that JA inhibits expression of WRKY70 and PR1 by both COI1-dependent and COI1-independent pathways.

[Detection of two viruses infecting Pinellia ternata in China].

OBJECTIVE: To study viruses infecting Pinellia ternata in China. METHOD: Symptom observation, DAS-ELISA and RT-PCR detection were applied. RESULT AND CONCLUSION: During a survey in early spring, SMV and CMV were both commonly distributed as main viruses infecting P. ternata collected from different areas in China. But DsMV was the virus which infected P. ternate in natural condition. The infection ratio of cultivated P. ternate by SMV and CMV were 71.4% and 14.3% respectively for 21 samples collected from Ningbo, Zhejiang province; 100% and 44.4% for 18 samples from Xiaoshan, Zhejiang province; 61.9% and 33.3% for 21 samples from Hebei province; 50.0% and 41.7% for 12 samples from Anhui province; 16.7% and 16.7% for 12 samples from Sichuan province; 31.3% and none for 16 samples from Beijing. And the infection ratio of 25 wild samples from different areas of China infected by SMV and CMV were both 20.0%.

[Cloning and expression of the cry1Ac-tchiB fusion gene from Bacillus thuringinesis and Tobacco and its insecticidal synergistic effect].

A new fusion gene cry1Ac-tchiB was constructed to enhance the toxicity of crystal proteins, the cry1Ac gene of Bacillus thuringiensis strain 4.0718 was combined with a tchiB (deleted signal peptide and Enterokinase site sequence). In this process, the Enterokinase site sequence was inserted into the midst of these two genes. Then the combined fragment carrying the upstream promoter region and the downstream terminator region of cry1Ac gene were cloned into the shuttle vector pHT315. And after a series of enzyme digestions and subclonings two new expression vector pHUAccB6 and pHUAccB7 were obtained. The two vectors were transformed into B. thuringiensis acrystalliferous strain XBU001 with electroporation to obtain the recombinant strain HAccB6 and HAccB7. The fusion gene was expressed and the expression product was detected by SDS-PAGE. The result indicated that the recombinant Cry1Ac-tchiB protein accumulated to the level of 61.38% of total bacterial proteins. Cry1Ac protein accumulated to the level of 42% of total bacterial proteins. Chitinase activities is 5.2 time more than that of the control strain. Under Atomic Force Microscopy and SEM of crystals protein, there were some bipy ramidal crystals with a size of 1.5 x 3.0 microm. Bioassay showed that the fusion crystals from recombinant strain HAccB6 and HAccB7 were high toxic against third-instar larvae of Helicourpa armigora with the LC50 (after 72h) value of 9.10 micro/mL and 11.34 micro/mL.The constructed fusion proteins had more toxicity than Cry1Ac crystal proteins. The study will enhances the toxicity of B. thuringiensis Cry toxins protein and makes a ground for constructing the fusion genes of B. thuringiensis cry gene and other foreign toxin genes and the recombinant strain with high toxicity.

[Cloning and superexpression of cry1Ac gene from 20kb DNA associated with Bacillus thuringiensis Cry1A Crystal Protein].

The CrylA Crystal Protein from Bacillus thuringiensis is associated with DNA, but the role and sequences of these DNA molecules are unknown. CrylA bipyramidal crystals from B. thuringiensis strain 4.0718 was selectively dissolved and associated DNA was extracted from protoxin. The DNA was digested with Nde I to obtain 3 to 5 kb fragments and then the fragments were subcloned into pMD18-T vector, screening of recombinants were done by PCR-RFLP and sequencing. The ORF of cry1Ac gene was amplified by primers designed and then subcloned. The 3.5 kb BamH I and Sal I fragments of pMDX35 was inserted into the pET30a vector, giving 8.9 kb recombinant plasmid, pETX35. ETX35 strain were obtained by transformed pETX35 into B121 (DE3). A 141 kD fusion protein was superexpressed as inclusion bodies. Quantitative protein analysis indicated that the amount of 141 kD protein was above the level of 51.36% of total cellular protein. Plasmid pHTX42 constructed from shuttle vector pHT304 was transformed B. thuringiensis acrystalliferous strain XBU001 with electroporation to obtain the recombinant HTX42. The recombinant protein was found with a molecular mass of 130 kD on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Scanning analysis indicated that the expressed protein accounted up to 79.28% of total cellular proteins and accumulated in the cells mounted up to 64.13% of cellular dry weight. Under Atomic Force Microscopy (AFM), typical bipyramidal crystals from HTX42 strain were found with a size of 1.2 microm x 2.0 microm. Bioassay showed that these inclusion bodies of ETX35 strain and crystals from HTX42 strain were highly toxic against the larvae of Plutella xylostella. On such a base, constructing insecticidal recombinant and analyzing the source, structure, and function of the 20 kb DNA can be further achieved.