Hrip1, a novel protein elicitor from necrotrophic fungus, Alternaria tenuissima, elicits cell death, expression of defence-related genes and systemic acquired resistance in tobacco.

Here, we report the identification, purification, characterization and gene cloning of a novel hypersensitive response inducing protein secreted by necrotrophic fungus, Alternaria tenuissima, designated as hypersensitive response inducing protein 1 (Hrip1). The protein caused the formation of necrotic lesions that mimic a typical hypersensitive response and apoptosis-related events including DNA laddering. The protein-encoding gene was cloned by rapid amplification of cDNA ends (RACE) method. The sequence analysis revealed that the cDNA is 495 bp in length and the open reading frame (ORF) encodes for a polypeptide of 163 amino acids with theoretical pI of 5.50 and molecular weight of 17 562.5 Da. Hrip1 induced calcium influx, medium alkalinization, activation of salicylic acid-induced protein kinase and several defence-related genes after infiltration in tobacco leaves. Cellular damage, restricted to the infiltrated zone, occurred only several hours later, at a time when expression of defence-related genes was activated. After several days, systemic acquired resistance was also induced. The tobacco plant cells that perceived the Hrip1 generated a cascade of signals acting at local, short, and long distances, and caused the coordinated expression of specific defence responses in a way similar to hypersensitivity to tobacco mosaic virus. Thus, Hrip1 represents a powerful tool to investigate further the signals and their transduction pathways involved in induced disease resistance in necrotrophic fungi.

Cloning and sequence analysis of two banana bunchy top virus genomes in Hainan.

The genome of Banana bunchy top virus (BBTV) consists of six segments of single-stranded DNA of approximately 1 kb in length. We identified and sequenced the complete genomes of two BBTV isolates, one with and one without satellite DNA, from Haikou, Hainan, China. The Haikou-2 isolate contains six genomic segments and an additional satellite DNA while the Haikou-4 isolate contains only six genomic segments. Typical of other babuviruses, each genomic segment encodes a single open reading frame and contains the highly conserved stem-loop and major common regions. Phylogenetic analysis of the two Haikou isolates together with existing sequence records in GenBank confirmed the grouping of BBTV into two large groups and further refined the geographical distribution of each group. To accommodate the changes in the BBTV geographical distribution, the two groups are proposed as the Southeast Asian group and the Pacific-Indian Oceans group. Both the Haikou-2 and Haikou-4 isolates belong to the newly proposed Southeast Asian group.

Silencing of molt-regulating transcription factor gene, CiHR3, affects growth and development of sugarcane stem borer, Chilo infuscatellus.

RNA interference (RNAi) is a technology for conducting functional genomic studies and a potential tool for crop protection against insect pests. Development of reliable methods for production and delivery of double-stranded RNA (dsRNA) is the major challenge for efficient pest control. In this study, Chilo infuscatellus Snellen (Crambidae: Lepidoptera) was fed with CiHR3 dsRNA expressed in bacteria or synthesized in vitro. The dsRNA ingested by C. infuscatellus successfully triggered silencing of the molt-regulating transcription factor CiHR3, an important gene for insect growth and development, and caused significant abnormalities and weight loss in insects within seven days of treatment. This study is an ideal example of feeding-based RNAi mediated by dsRNA expressed in bacteria or synthesized in vitro. The results also suggested that feeding-based RNA interference is a potential method for the management of C. infuscatellus.

Identification, characterization, and expression of a novel P450 gene encoding CYP6AE25 from the Asian corn borer, Ostrinia furnacalis.

An allele of the cytochrome P450 gene, CYP6AE14, named CYP6AE25 (GenBank accession no. EU807990) was isolated from the Asian com borer, Ostrinia fumacalis (Guenée) (Lepidoptera: Pyralidae) by RT-PCR. The cDNA sequence of CYP6AE25 is 2315 bp in length and contains a 1569 nucleotides open reading frame encoding a putative protein with 523 amino acid residues and a predicted molecular weight of 59.95 kDa and a theoretical pI of 8.31. The putative protein contains the classic heme-binding sequence motif F××G×××C×G (residues 451-460) conserved among all P450 enzymes as well as other characteristic motifs of all cytochrome P450s. It shares 52% identity with the previously published sequence of CYP6AE14 (GenBank accession no. DQ986461) from Helicoverpa armigera. Phylogenetic analysis of amino acid sequences from members of various P450 families indicated that CYP6AE25 has a closer phylogenetic relationship with CYP6AE14 and CYP6B1 that are related to metabolism of plant allelochemicals, CYP6D1 which is related to pyrethroid resistance and has a more distant relationship to CYP302A1 and CYP307A1 which are related to synthesis of the insect molting hormones. The expression level of the gene in the adults and immature stages of O. furnacalis by quantitative real-time PCR revealed that CYP6AE25 was expressed in all life stages investigated. The mRNA expression level in 3(rd) instar larvae was 12.8- and 2.97-fold higher than those in pupae and adults, respectively. The tissue specific expression level of CYP6AE25 was in the order of midgut, malpighian tube and fatty body from high to low but was absent in ovary and brain. The analysis of the CYP6AB25 gene using bioinformatic software is discussed.

Baseline Susceptibility of Spodoptera frugiperda Populations Collected in India towards Different Chemical Classes of Insecticides.

Fall armyworm (FAW), Spodoptera frugiperda, is a major pest of maize in the Americas and recently invaded the Eastern hemisphere. It was first detected in India in 2018 and is considered a major threat to maize production. FAW control largely relies on the application of chemical insecticides and transgenic crops expressing Bacillus thuringiensis insecticidal proteins. Assessing FAW resistance and insecticide susceptibility is a cornerstone to develop sustainable resistance management strategies. In this study, we conducted more than 400 bioassays to assess the efficacy of nine insecticides from seven mode-of-action classes against 47 FAW populations collected in 2019 and 2020 across various geographical areas in India. The resistance status of the field-collected populations was compared to an Indian population sampled in 2018, and an insecticide susceptible reference population collected in 2005 in Brazil. Low to moderate resistance levels were observed for thiodicarb, chlorpyriphos, deltamethrin, chlorantraniliprole and flubendiamide in several populations (including the reference population collected in 2018). The highest resistance ratios were observed for deltamethrin which likely compromises recommended label rates for pyrethroid insecticides in general. Our data provide a useful baseline for future FAW resistance monitoring initiatives and highlight the need to implement insecticide resistance management strategies.