Ostrinia furnacalis, is the major pest of maize causing significant yield losses. So far, many approaches have been used to increase the virulence of entomopathogenic fungal isolates. The current study is an attempt to estimate synergistic effect of Beauveria bassiana and Trichoderma asperellum in order to explore larval immune response through RNA sequencing and differentially expression analysis. In vivo synergism was examined in seven proportions (B. bassiana: T. asperellum = 1:1, 1:2, 1:3, 1:4, 4:1, 3:1, 2:1) and in the in vitro case, two inoculation methods were applied: seed coating and soil drenching. Results revealed significant decrease in plant damage and high larval mortality in fungal treatments. Fungal isolates mediated the plant defense by increasing proline, superoxide dismutase (SOD), peroxidase (POD), polyphenol oxidase (PPO) and protease activities. Seed coating method was proved to be the most effective in case of maize endophytic colonization. In total, 59 immune-related differentially expressed genes DEGs were identified including, cytochrome P450, heat shock protein, ABC transporter, cadherin, peptidoglycan recognition protein (PGRP), cuticlular protein, etc. Further, transcriptomic response was confirmed by qRT-PCR. Our results concluded that, coculture of B. bassiana and T. asperellum has the synergistic potential to suppress the immune response of O. furnacalis and can be used as sustainable approach to induce plant resistance through activation of defense-related enzymes.

WRKY transcription factors comprise one of the largest gene families and serve as key regulators of plant defenses against herbivore attack. However, studies related to the roles of WRKY genes in response to herbivory are limited in maize. In this study, a total of 128 putative maize WRKY genes (ZmWRKYs) were identified from the new maize genome (v4). These genes were divided into seven subgroups (groups I, IIa–e, and III) based on phylogenomic analysis, with distinct motif compositions in each subgroup. Syntenic analysis revealed that 72 (56.3%) of the genes were derived from either segmental or tandem duplication events (69 and 3, respectively), suggesting a pivotal role of segmental duplication in the expansion of the ZmWRKY family. Importantly, transcriptional regulation prediction showed that six key WRKY genes contribute to four major defense-related pathways: L-phenylalanine biosynthesis II and flavonoid, benzoxazinoid, and jasmonic acid (JA) biosynthesis. These key WRKY genes were strongly induced in commercial maize (Jingke968) infested with the Asian corn borer, Ostrinia furnacalis, for 0, 2, 4, 12 and 24 h in the field, and their expression levels were highly correlated with predicted target genes, suggesting that these genes have important functions in the response to O. furnacalis. Our results provide a comprehensive understanding of the WRKY gene family based on the new assembly of the maize genome and lay the foundation for further studies into functional characteristics of ZmWRKY genes in commercial maize defenses against O. furnacalis in the field.

Two colonies of Asian corn borer, Ostrinia furnacalis (Guenée), artificially selected from a Bt-susceptible colony (ACB-BtS) for resistance to Cry1Ab (ACB-AbR) and Cry1Ac (ACB-AcR) toxins, were used to analyze inheritance patterns of resistance to Cry1 toxins. ACB-AbR and ACB-AcR evolved significant levels of resistance, with resistance ratios (RR) of 39-fold and 78.8-fold to Cry1Ab and Cry1Ac, respectively. The susceptibility of ACB-AbR larvae to Cry1Ac and Cry1F toxins, which had not previously been exposed, were significantly reduced, being >113-fold and 48-fold, respectively. Similarly, susceptibility of ACB-AcR larvae to Cry1Ab and Cry1F were also significantly reduced (RR > nine-fold, RR > 18-fold, respectively), indicating cross-resistance among Cry1Ab, Cry1Ac, and Cry1F toxins. However, ACB-AbR and ACB-AcR larvae were equally susceptible to Cry1Ie as were ACB-BtS larvae, indicating no cross-resistance between Cry1Ie and Cry1Ab or Cry1Ac toxins; this may provide considerable benefits in preventing or delaying the evolution of resistance in ACB to Cry1Ab and Cry1Ac toxins. Backcrossing studies indicated that resistance to Cry1Ab toxin was polygenic in ACB-AbR, but monogenic in ACB-AcR, whilst resistance to Cry1Ac toxin was primarily monogenic in both ACB-AbR and ACB-AcR, but polygenic as resistance increased.

Asian corn borer (ACB), Ostrinia furnacalis (Guenée), is the major insect pest of maize in China and countries of East and Southeast Asia, the Pacific and Australasia. ACB can develop strong resistance to the transgenic Bt maize expressing Cry1Ab, the most widely commercialized Bt maize worldwide. However, the molecular basis for the resistance mechanisms of ACB to Cry1Ab remained unclear. Two biological replicates of the transcriptome of Bt susceptible (ACB-BtS) and Cry1Ab resistant (ACB-AbR) strains of ACB were sequenced using Solexa/Illumina RNA-Seq technology to identify Cry1Ab resistance-relevant genes. The numbers of unigenes for two biological replications were 63,032 and 53,710 for ACB-BtS and 57,770 and 54,468 for ACB-AbR. There were 35,723 annotated unigenes from ACB reads found by BLAST searching NCBI non-redundant, NCBI non-redundant nucleotide, Swiss-prot protein, Kyoto Encyclopedia of Genes and Genomes, Cluster of Orthologous Groups of proteins, and Gene Ontology databases. Based on the NOISeq method, 3,793 unigenes were judged to be differentially expressed between ACB-BtS and ACB-AbR. Cry1Ab resistance appeared to be associated with change in the transcription level of enzymes involved in growth regulation, detoxification and metabolic/catabolic process. Among previously described Bt toxin receptors, the differentially expressed unigenes associated with aminopeptidase N and chymotrypsin/trypsin were up-regulated in ACB-AbR. Whereas, other putative Cry receptors, cadherin-like protein, alkaline phosphatase, glycolipid, actin, V-type proton ATPase vatalytic, heat shock protein, were under-transcripted. Finally, GPI-anchor biosynthesis was found to be involved in the significantly enriched pathway, and all genes mapped to the pathway were substantially down-regulated in ACB-AbR. To our knowledge, this is the first comparative transcriptome study to discover candidate genes involved in ACB Bt resistance. This study identified differentially expressed unigenes related to general Bt resistance in ACB. The assembled, annotated transcriptomes provides a valuable genomic resource for further understanding of the molecular basis of ACB Bt resistance mechanisms.

Development of resistance in target pests is a major threat to long-term use of transgenic crops expressing Bacillus thuringiensis (Bt) Cry toxins. To manage and/or delay the evolution of resistance in target insects through the implementation of effective strategies, it is essential to understand the basis of resistance. One of the most important mechanisms of insect resistance to Bt crops is the alteration of the interactions between Cry toxins and their receptors in the midgut. A Cry1Ac-selected strain of Asian corn borer (ACB), Ostrinia furnacalis, a key pest of maize in China, evolved three mutant alleles of a cadherin-like protein (OfCAD) (MPR-r1, MPR-r2 and MPR-r3), which mapped within the toxin-binding region (TBR). Each of the three mutant alleles possessed two or three amino acid substitutions in this region, especially Thr¹⁴⁵⁷→Ser. In highly resistant larvae (ACB-Ac200), MPR-r2 had a 26-amino acid residue deletion in the TBR, which resulted in reduced binding of Cry1Ac compared to the MPR from the susceptible strain, suggesting that the number of amino acid deletions influences the level of resistance. Furthermore, downregulation of OfCAD gene (ofcad) transcription was observed in the Cry1Ac resistant strain, ACB-Ac24, suggesting that Cry1Ac resistance in ACB is associated with the downregulation of the transcript levels of the cadherin-like protein gene. The OfCAD identified from ACB exhibited a high degree of similarity to other members of the cadherin super-family in lepidopteran species.

Transgenic crops producing insecticidal proteins are effective to manage lepidopteran pests. Development of insect-resistance is the major threat to Bacillus thuringiensis (Bt) crops such as Cry1Ah-Maize. Laboratory selection with Bt-Cry1Ah toxin incorporated in artificial diet, during 48 generations of Asian corn borer (ACB) Ostrinia furnacalis produced 200-fold resistance. This resistant colony ACB-AhR readily consumed and survived on Cry1Ah-expressing Bt-maize. Cross-resistance analysis showed high cross-resistance to Cry1F (464-fold), moderate cross-resistance to Cry1Ab (28.38-fold), Cry1Ac (22.11-fold) and no cross-resistance to Cry1Ie toxin. This ACB-AhR cross-resistant phenotype is different from ACB-Cry1Fa resistant population that showed no cross resistance to Cry1Ah, suggesting that different mechanisms of resistance were selected in these two populations. Bioassays of reciprocal F₁ crosses-progeny suggested autosomal inheritance of Cry1Ah resistance with no maternal effects. The dominance of resistance increased as concentration decreased. In Cry1Ah-maize tissues the progeny of reciprocal F₁ crosses behaved as functionally recessive. Progenies analysis from backcrosses (F_1 ×resistant strain) suggested polygenic contribution to Cry1Ah- resistance in ACB-AhR. The use of multiple toxins is an imperative factor for delaying evolution of resistance to Cry1Ah-corn in ACB. However, the fact that ACB-AhR showed cross resistance to Cry1Fa indicates that selection of toxins for pyramided plants should be carefully done.