RNA interference (RNAi) caused by exogenous double-stranded RNA (dsRNA) has developed into a powerful technique in functional genomics, and to date it is widely used to down-regulate crucial physiology-related genes to control pest insects. A molt-regulating transcription factor gene, HaHR3, of cotton bollworm (Helicoverpa armigera) was selected as the target gene. Four different fragments covering the coding sequence (CDS) of HaHR3 were cloned into vector L4440 to express dsRNAs in Escherichia coli. The most effective silencing fragment was then cloned into a plant over-expression vector to express a hairpin RNA (hpRNA) in transgenic tobacco (Nicotiana tabacum). When H. armigera larvae were fed the E. coli or transgenic plants, the HaHR3 mRNA and protein levels dramatically decreased, resulting developmental deformity and larval lethality. The results demonstrate that both recombinant bacteria and transgenic plants could induce HaHR3 silence to disrupt H. armigera development, transgenic plant-mediated RNAi is emerging as a powerful approach for controlling insect pests.

Verticillium dahliae is a wide-host-range fungal pathogen that causes soil-borne disease in hundreds of dicotyledonous hosts. In search of V. dahliae Vd991 cell death-inducing proteins, we identified a pectate lyase (VdPEL1) that exhibited pectin hydrolytic activity, which could induce strong cell death in several plants. Purified VdPEL1 triggered defense responses and conferred resistance to Botrytis cinerea and V. dahliae in tobacco and cotton plants. Our results demonstrated that the mutant VdPEL1^rec lacking the enzymatic activity lacked functions to induce both cell death and plant resistance, implying that the enzymatic activity was necessary. In addition, VdPEL1 was strongly induced in V. dahliae infected Nicotiana benthamiana and cotton roots, and VdPEL1 deletion strains severely compromised the virulence of V. dahliae. Our data suggested that VdPEL1 contributed to V. dahliae virulence and induced plant defense responses. These findings provide a new insight for the function of pectate lyase in the host–pathogen interaction.

Botrytis cinerea is one of the most notorious pathogenic species that causes serious plant diseases and substantial losses in agriculture throughout the world. We identified BcXyl1 from B. cinerea that exhibited xylanase activity. Expression of the BcXyl1 gene was strongly induced in B. cinerea infecting Nicotiana benthamiana and tomato plants, and BcXyl1 deletion strains severely compromised the virulence of B. cinerea. BcXyl1 induced strong cell death in several plants, and cell death activity of BcXyl1 was independent of its xylanase activity. Purified BcXyl1 triggered typically PAMP-triggered immunity (PTI) responses and conferred resistance to B. cinerea and TMV in tobacco and tomato plants. A 26-amino acid peptide of BcXyl1 was sufficient for elicitor function. Furthermore, the BcXyl1 death-inducing signal was mediated by the plant LRR receptor-like kinases (RLKs) BAK1 and SOBIR1. Our data suggested that BcXyl1 contributed to B. cinerea virulence and induced plant defense responses.

Alt a 1 family proteins (AA1s) have only been observed in the Dothideomycetes and Sordariomycetes classes of fungi, and their biological functions have remained poorly understood. Verticillium dahliae, a soil-borne pathogen that causes plant wilt disease, secretes hundreds of proteins during the process of pathogenic infection, including the AA1 member PevD1. In this study, we found that the pevd1 transcript was present in all of the hosts studied (cotton, Arabidopsis, tomato, and tobacco) and showed elevated expression throughout the infection process. Furthermore, pevd1 knockout mutants displayed attenuated pathogenicity compared with the wild-type (WT) strain and complemented strains in hosts. A partner protein of PevD1, pathogenesis-related protein 5 (PR5)-like protein GhPR5, was isolated from cotton (Gossypium hirsutum) plants by co-purification assays, and the PevD1–GhPR5 interaction was determined to be localized in the C-terminus (PevD1b, amino acids residues 113–155) by pull-down and yeast two-hybrid techniques. Re-introduction of the pevd1b gene into a pevd1 knockout mutant resulted in restoration of the virulence phenotype to WT levels. In addition, PevD1b, which is similar to PevD1, decreased the antifungal activity of GhPR5 in vitro. Our findings reveal an infection strategy in which V. dahliae secretes PevD1 to inhibit GhPR5 antifungal activity in order to overcome the host defence system.

Here we reported a novel protein elicitor from Bacillus amyloliquefaciens NC6 induced systemic resistance (ISR) in tobacco. The purification was executed by ion-exchange chromatography, native-page extraction and HPLC, and the amino acid sequence was identified by mass spectrometry. This recombinant elicitor protein, expressed in Escherichia coli by an E1 expression vector, had good thermal stability, and the elicitor caused a clearly defined hypersensitive response (HR) necrosis in tobacco leaves. It could also trigger early defence events, including generation of reactive oxygen species (H₂O₂ and O₂^-) and phenolic-compound accumulation. Quantitative real-time PCR (Q-RT-PCR) results indicated that several plant defence genes, including the salicylic acid (SA)-responsive PR1a, PR1b, PR5, and phenylalanine ammonia lyase (PAL), as well as the jasmonic acid (JA)-responsive PDF1.2 and CORONATINE INSENSITIVE 1 (COI1), were all up-regulated. Moreover, infiltration conferred systemic resistance against a broad spectrum of pathogens, including Tobacco mosaic virus (TMV) and the fungal pathogen Botrytis cinerea.

During pathogenic infection, hundreds of proteins that play vital roles in the Verticillium dahliae-host interaction are secreted. In this study, an integrated proteomic analysis of secreted V. dahliae proteins was performed, and a conserved secretory protein, designated VdCP1, was identified as a member of the SnodProt1 phytotoxin family. An expression analysis of the vdcp1 gene revealed that the transcript is present in every condition studied and displays elevated expression throughout the infection process. To investigate the natural role of VdCP1 in V. dahliae, two vdcp1 knockout mutants and their complementation strains were generated. Bioassays of these mutants revealed no obvious phenotypic differences from the wild-type (WT) in terms of mycelial growth, conidial production or mycelial/spore morphology. However, compared with the WT, the vdcp1 knockout mutants displayed attenuated pathogenicity in cotton plants. Furthermore, treating plants with purified recombinant VdCP1 protein expressed in Pichia pastoris induced the accumulation of reactive oxygen species (ROS), expression of several defense-related genes, leakage of ion electrolytes, enhancement of defense-related enzyme activity and production of salicylic acid. Moreover, VdCP1 conferred resistance to Botrytis cinerea and Pseudomonas syringae pv. tabaci in tobacco and to V. dahliae in cotton. Further research revealed that VdCP1 possesses chitin-binding properties and that the growth of vdcp1 knockout mutants was more affected by treatments with chitinase, indicating that VdCP1 could protect V. dahliae cell wall from enzymatic degradation, which suggests an effector role of VdCP1 in infecting hosts.