Clubroot caused by Plasmodiophora brassicaeis one of the most important diseases in cruciferous crops. The recognition of P. brassicae by host plants is thought to occur at the primary infection stage, but the underlying mechanism remains unclear. Secretory proteins as effector candidates play critical roles in the recognition of pathogens and the interactions between pathogens and hosts. In this study, 33 P. brassicae secretory proteins expressed during primary infection were identified through transcriptome, secretory protein prediction, and yeast signal sequence trap analyses. Furthermore, the proteins that could suppress or induce cell death were screened through an Agrobacterium-mediated plant virus transient expression system and a protoplast transient expression system. Two secretory proteins, PBCN_002550 and PBCN_005499, were found to be capable of inducing cell death associated with H₂O₂ accumulation and electrolyte leakage in Nicotiana benthamiana. Moreover, PBCN_002550 could also induce cell death in Chinese cabbage. In addition, 24 of the remaining 31 tested secretory proteins could suppress mouse Bcl-2-associated X protein-induced cell death, and 28 proteins could suppress PBCN_002550-induced cell death.

Phenylalanine ammonia-lyase (PAL) is a key enzyme in the phenylalanine metabolism pathway and plays an important role in plant growth and stress response. It has been widely reported in plants, but less studied in wheat. In this study, 54 PAL genes were identified in the wheat genome. Based on phylogenetic analysis, the 54 TaPAL genes were divided into four groups (I, II, III, and IV). Then, the expression levels of TaPALs under biotic stresses were analyzed by transcriptome data analysis. The results showed that 31 genes were up-regulated and one gene was down-regulated after inoculation with Fusarium graminearum, 11 genes were up-regulated and 14 genes were down-regulated after inoculation with Puccinia striiformis, and 32 up-regulated and three down-regulated genes after inoculation with powdery mildew. The expression patterns of the five TaPALs were further analyzed by qRT-PCR. After inoculation with F. graminearum, the expression levels of five TaPALs were up-regulated. However, the TaPALs (expect TaPAL49) were down-regulated when inoculated with P. striiformis. Finally, the functions of TaPAL32 and TaPAL42 in resistance of wheat to the stripe rust were further analyzed by virus induced gene silencing (VIGS) assays. The results showed that the disease severity of TaPAL32 and TaPAL42 silenced plants was higher than that of control plants at 14 days after inoculation. It indicated that these two genes played a positive role in wheat stripe rust resistance. This study provided new evidence support for the functional study of PAL genes in wheat, and provided potential application value for the breeding of wheat resistant varieties.

ZF-HD family genes play important roles in plant growth and development. Studies about the whole genome analysis of ZF-HD gene family have been reported in some plant species. In this study, the whole genome identification and expression profile of the ZF-HD gene family were analyzed for the first time in wheat. A total of 37 TaZF-HD genes were identified and divided into TaMIF and TaZHD subfamilies according to the conserved domain. The phylogeny tree of the TaZF-HD proteins was further divided into six groups based on the phylogenetic relationship. The 37 TaZF-HDs were distributed on 18 of 21 chromosomes, and almost all the genes had no introns. Gene duplication and Ka/Ks analysis showed that the gene family may have experienced powerful purification selection pressure during wheat evolution. The qRT-PCR analysis showed that TaZF-HD genes had significant expression patterns in different biotic stress and abiotic stress. Through subcellular localization experiments, we found that TaZHD6-3B was located in the nucleus, while TaMIF4-5D was located in the cell membrane and nucleus. Our research contributes to a comprehensive understanding of the TaZF-HD family, provides a new perspective for further research on the biological functions of TaZF-HD genes in wheat.

Brassica napus (oilseed rape) is an economically important oil crop worldwide. Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum is a threat to oilseed rape production. Because the flower petals play pivotal roles in the SSR disease cycle, it is useful to express the resistance-related genes specifically in flowers to hinder further infection with S. sclerotiorum. To screen flower-specific promoters, we first analyzed the transcriptome data from 12 different tissues of the B. napus line ZS11. In total, 249 flower-specific candidate genes with high expression in petals were identified, and the expression patterns of 30 candidate genes were verified by quantitative real-time transcription-PCR (qRT-PCR) analysis. Furthermore, two novel flower-specific promoters (FSP046 and FSP061 promoter) were identified, and the tissue specificity and continuous expression in petals were determined in transgenic Arabidopsis thaliana with fusing the promoters to β-glucuronidase (GUS)-reporter gene. GUS staining, transcript expression pattern, and GUS activity analysis indicated that FSP046 and FSP061 promoter were strictly flower-specific promoters, and FSP046 promoter had a stronger activity. The two promoters were further confirmed to be able to direct GUS expression in B. napus flowers using transient expression system. The transcriptome data and the flower-specific promoters screened in the present study will benefit fundamental research for improving the agronomic traits as well as disease and pest control in a tissue-specific manner.

Sucrose non-fermenting-1-related protein kinases (SnRKs) play vital roles in plant growth and stress responses. However, little is known about the SnRK functions in wheat. In this study, 149 TaSnRKs (wheat SnRKs) were identified and were divided into three subfamilies. A combination of public transcriptome data and real-time reverse transcription-polymerase chain reaction (qRT-PCR) analysis revealed the distinct expression patterns of TaSnRKs under various abiotic and biotic stresses. TaSnRK2.4-B, a member of SnRK2s, has different expression patterns under polyethylene glycol (PEG), sodium chloride (NaCl) treatment, and high concentrations of abscisic acid (ABA) application. Yeast two-hybrid assay indicated that TaSnRK2.4-B could interact with the SnRK2-interacting calcium sensor (SCS) in wheat and play a role in the ABA-dependent pathway. Moreover, TaSnRK2.4-B might be a negative regulator in wheat against pathogen infection. The present study provides valuable information for understanding the functions of the TaSnRK family and provides recommendations for future genetic improvement in wheat stress resistance.

Sharp eyespot is a crucial disease affecting cereal plants, such as bread wheat (Triticum aestivum) and barley (Hordeum vulgare), and is primarily caused by the pathogenic fungus Rhizoctonia cerealis. As disease severity has increased, it has become imperative to find an effective and reasonable control strategy. One such strategy is the use of the trehalose analog, validamycin, which has been shown to have a potent inhibitory effect on several trehalases found in both insects and fungi, and is widely used as a fungicide in agriculture. In this study, we demonstrated that 0.5 μg/mL validamycin on PDA plates had an inhibitory effect on R. cerealis strain R0301, but had no significant impact on Fusarium graminearum strain PH-1. Except for its inhibiting the trehalase activity of pathogenic fungi, little is known about its mechanism of action. Six trehalase genes were identified in the genome of R. cerealis, including one neutral trehalase and five acidic trehalase genes. Enzyme activity assays indicated that treatment with 5 μg/mL validamycin significantly reduces trehalase activity, providing evidence that validamycin treatment does indeed affect trehalase, even though the expression levels of most trehalase genes, except Rc17406, were not obviously affected. Transcriptome analysis revealed that treatment with validamycin downregulated genes involved in metabolic processes, ribosome biogenesis, and pathogenicity in the R. cerealis. KEGG pathway analysis further showed that validamycin affected genes related to the MAPK signaling pathway, with a significant decrease in ribosome synthesis and assembly. In conclusion, our results indicated that validamycin not only inhibits trehalose activity, but also affects the ribosome synthesis and MAPK pathways of R. cerealis, leading to the suppression of fungal growth and pesticidal effects. This study provides novel insights into the mechanism of action of validamycin.