Ke Duan

Genome-wide identification and comparative expression analysis of NBS–LRR-encoding genes upon Colletotrichum gloeosporioides infection in two ecotypes of Fragaria vesca

Anthracnose caused by Colletotrichum spp. is one of the most destructive diseases of cultivated strawberry (Fragaria×ananassa Duchesne) worldwide. The correlation between NBS-LRR genes, the largest class of known resistance genes, and strawberry anthracnose resistance has been elusive. BLAST search in NCBI identified 94 FvNBSs in the diploid genome of strawberry Fragaria vesca, with 67 of the TIR-NBS-LRR type. At least 36 FvNBSs were expressed, with 25% being non-coding genes. Two F. vesca ecotypes, HLJ and YW, showed great variations in both morphological and physiological responses upon C. gloeosporioides infection. qRT-PCR revealed that 5 of the 12 leaf-expressed FvNBSs displaying opposite transcription responses to C. gloeosporioides infection in two ecotypes. These results showed that the transcriptional responses of several FvNBSs were involved in the ecotype-specific responses to C. gloeosporioides in F. vesca. These FvNBSs hold potential in characterizing molecular components and developing novel markers associated with anthracnose resistance in strawberry.

Auxin Biosynthesis by the YUCCA6 Flavin Monooxygenase Gene in Woodland Strawberry

Auxin has been regarded as the main signal molecule coordinating the growth and ripening of fruits in strawberry, the reference genomic system for Rosaceae. The mechanisms regulating auxin biosynthesis in strawberry are largely elusive. Recently, we demonstrated that two YUCCA genes are involved in flower and fruit development in cultivated strawberry. Here, we show that the woodland strawberry (Fragaria vesca L.) genome harbors nine loci for YUCCA genes and eight of them encode functional proteins. Transcription pattern in different plant organs was different for all eight FvYUCs. Functionality of the FvYUC6 gene was studied in transgenic strawberry overexpressing FvYUC6, which showed typical high-auxin phenotypes. Overexpression of FvYUC6 also delayed flowering and led to complete male sterility in F. vesca. Additionally, specific repression of FvYUC6 expression by RNA interference significantly inhibited vegetative growth and reduced plant fertility. The development of leaves, roots, flowers, and fruits was greatly affected in FvYUC6-repressed plants. Expression of a subset of auxin-responsive genes was well correlated with the changes of FvYUC6 transcript levels and free indole-3-acetic acid levels in transgenic strawberry. These observations are consistent with an important role of FvYUC6 in auxin synthesis, and support a main role of the gene product in vegetative and reproductive development in woodland strawberry.

The different interactions of Colletotrichum gloeosporioides with two strawberry varieties and the involvement of salicylic acid

The disease symptoms recognized as ‘Anthracnose’ are caused by Colletotrichum spp. and lead to large-scale strawberry (Fragaria×ananassa Duchesne) losses worldwide in terms of both quality and production. Little is known regarding the mechanisms underlying the genetic variations in the strawberry–Colletotrichum spp. interaction. In this work, Colletotrichum gloeosporioides (C. gloeosporioides) infection was characterized in two varieties exhibiting different susceptibilities, and the involvement of salicylic acid (SA) was examined. Light microscopic observation showed that C. gloeosporioides conidia germinated earlier and faster on the leaf surface of the susceptible cultivar compared with the less-susceptible cultivar. Several PR genes were differentially expressed, with higher-amplitude changes observed in the less-susceptible cultivar. The less-susceptible cultivar contained a higher level of basal SA, and the SA levels increased rapidly upon infection, followed by a sharp decrease before the necrotrophic phase. External SA pretreatment reduced susceptibility and elevated the internal SA levels in both varieties, which were sharply reduced in the susceptible cultivar upon inoculation. The less-susceptible cultivar also displayed a more sensitive and marked increase in the transcripts of NB-LRR genes to C. gloeosporioides, and SA pretreatment differentially induced transcript accumulation in the two varieties during infection. Furthermore, SA directly inhibited the germination of C. gloeosporioides conidia; NB-LRR transcript accumulation in response to SA pretreatment was both dose- and cultivar-dependent. The results demonstrate that the less-susceptible cultivar showed reduced conidia germination. The contribution of SA might involve microbial isolate-specific sensitivity to SA, cultivar/tissue-specific SA homeostasis and signaling, and the sensitivity of R genes and the related defense network to SA and pathogens.

Novel Fungal Pathogenicity and Leaf Defense Strategies Are Revealed by Simultaneous Transcriptome Analysis of Colletotrichum fructicola and Strawberry Infected by This Fungus.

Colletotrichum fructicola, which is part of the C. gloeosporioides species complex, can cause anthracnose diseases in strawberries worldwide. However, the molecular interactions between C. fructicola and strawberry are largely unknown. A deep RNA-sequencing approach was applied to gain insights into the pathogenicity mechanisms of C. fructicola and the defense response of strawberry plants at different stages of infection. The transcriptome data showed stage-specific transcription accompanied by a step-by-step strawberry defense response and the evasion of this defense system by fungus. Fungal genes involved in plant cell wall degradation, secondary metabolism, and detoxification were up-regulated at different stage of infection. Most importantly, C. fructicola infection was accompanied by a large number of highly expressed effectors. Four new identified effectors function in the suppression of Bax-mediated programmed cell death. Strawberry utilizes pathogen-associated molecular patterns (PAMP)-triggered immunity and effector-triggered immunity to prevent C. fructicola invasion, followed by the initiation of downstream innate immunity. The up-regulation of genes related to salicylic acid provided evidence that salicylic acid signaling may serve as the core defense signaling mechanism, while jasmonic acid and ethylene pathways were largely inhibited by C. fructicola. The necrotrophic stage displayed a significant up-regulation of genes involved in reactive oxygen species activation. Collectively, the transcriptomic data of both C. fructicola and strawberry shows that even though plants build a multilayered defense against infection, C. fructicola employs a series of escape or antagonizing mechanisms to successfully infect host cells.

Identification of FaNBS-encoding genes responsive to Colletotrichum fructicola infection in strawberry (Fragaria ×ananassa Duchase)

Anthracnose caused by C. fructicola is a serious fungal disease in strawberry (Fragaria ×ananassa), but the molecular mechanism is still unclear. Here, approximately 232 FaNBS-encoding genes were identified by RNA-seq, of which 53 were specifically annotated in the Fragaria ×ananassa genome. After C. fructicola inoculation, a total of 12, 9 and 24 differentially expressed FaNBS-encoding genes were observed at 24, 72, and 96 Hpi in the Fragaria ×ananassa (cv. Jiuxiang). Furthermore, we manually annotated 40 FaNBS-encoding genes fused with other integrated domains, 27 of which are FaNBS-RPW8 fusions. Most of the RPW8 domains from the FaNBS-RPW8 fusions were conserved in the tyrosine (Y) and glycine (G) residues. Interestingly, the heatmap, Venn diagram and qRT-PCR analysis showed that the expression of an FaNBS-RPW8 fusion (mrna24117.1-v1.0-hybrid) was markedly induced at different stages after C. fructicola inoculation in the susceptible cultivar of Jiuxiang. Altogether, the results of the present study provide information from comprehensive structure and expression analyses of FaNBS-encoding genes and add insight into the molecular mechanism underlying FaNBS-RPW8-mediated resistance to C. fructicola in strawberry (Fragaria ×ananassa).