Nitrate reductase is required for sclerotial development and virulence of Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum, the causal agent of Sclerotinia stem rot (SSR) on more than 450 plant species, is a notorious fungal pathogen. Nitrate reductase (NR) is required for nitrate assimilation that mediates the reduction of nitrate to nitrite and is the major enzymatic source for NO production in fungi. To explore the possible effects of nitrate reductase SsNR on the development, stress response, and virulence of S. sclerotiorum, RNA interference (RNAi) of SsNR was performed. The results showed that SsNR-silenced mutants showed abnormity in mycelia growth, sclerotia formation, infection cushion formation, reduced virulence on rapeseed and soybean with decreased oxalic acid production. Furthermore SsNR-silenced mutants are more sensitive to abiotic stresses such as Congo Red, SDS, H2O2, and NaCl. Importantly, the expression levels of pathogenicity-related genes SsGgt1, SsSac1, and SsSmk3 are down-regulated in SsNR-silenced mutants, while SsCyp is up-regulated. In summary, phenotypic changes in the gene silenced mutants indicate that SsNR plays important roles in the mycelia growth, sclerotia development, stress response and fungal virulence of S. sclerotiorum.

Overexpression of AGAMOUS-like gene PfAG5 promotes early flowering in Polypogon fugax.

Herbicides are the major tool for controlling large populations of yield depleting weeds. However, over-reliance on herbicides has resulted in weed adaptation and herbicide resistance. In recent years, early flowering weed species related to herbicide resistance is emerging, which may cause seed loss before crop harvest, creating a new problem for non-chemical weed management. In this study, a homologue gene of AGAMOUS sub-family (referred to as PfAG5) of the MADS-box family was cloned from plants of an early flowering Polypogon fugax Nees ex Steud. population resistant to the ACCase inhibitor herbicide (clodinafop-propargyl). The PfAG5 gene was functionally characterised in Arabidopsis thaliana L. Overexpression of the PfAG5 gene in Arabidopsis resulted in early flowering, abnormal flowers (e.g. small petals), short plants and reduced seed set, compared with the wild type. The expression of the PfAG5 gene was high in leaves and flowers, but low in pods in transgenic Arabidopsis. The PfAG5 gene was expressed earlier and higher in the resistant (R) than the susceptible (S) P. fugax plants. Furthermore, one protein (FRIGIDA-like) with relevance to flowering time regulation and interacts with PfAG5 in resistant (R) P. fugax was identified by the yeast two-hybrid and pull-down assays. These results suggest that the PfAG5 gene is involved in modulating early flowering in P. fugax.

StMADS11 Subfamily Gene PfMADS16 From Polypogon fugax Regulates Early Flowering and Seed Development.

The evolution of herbicide resistance in weedy plants leads to various adaptation traits including flowering time and seed germination. In our previous studies, we found an association of the early flowering phenotype with the ACCase inhibitor herbicide resistance genotype in a population of Polypogon fugax. MADS-box transcription factors are known to play pivotal roles in regulating plant flowering time. In this study, a SHORT VEGETATIVE PHASE (SVP)-like gene, belonging to the StMADS11 subfamily in the MADS-box family, was cloned from the early flowering P. fugax population (referred to as PfMADS16) and resistant to the herbicide clodinafop- propargyl. Overexpression of the SVP-like gene PfMADS16 in Arabidopsis thaliana resulted in early flowering and seed abortion. This is consistent with the phenotypic characters of resistant P. fugax plants, but contrary to the conventional role of SVP-like genes that usually suppress flowering. In addition, down regulation of the seed formation gene AtKTN1 in flowers of PfMADS16 transgenic Arabidopsis plants indicates that PfMADS16 may be indirectly associated with seed viability. Furthermore, one protein (PfMADS2) from the APETALA1 (AP1) subfamily interacting with PfMADS16 in P. fugax was identified with relevance to flowering time regulation. These results suggest that the PfMADS16 gene is an early flowering regulation gene associated with seed formation and viability in resistant P. fugax population. Our study provides potential application of PfMADS16 for integrated weed management (such as genetic-based weed control strategies) aiming to reduce the soil weed seedbank.

SsSm1, a Cerato-platanin family protein, is involved in the hyphal development and pathogenic process of Sclerotinia sclerotiorum.

The filamentous fungus Sclerotinia sclerotiorum is an important plant pathogen with a worldwide distribution. It can infect a wide variety of plants, causing serious disease in many types of crops, such as rapeseed, sunflower and soybean. Sclerotinia stem rot caused by this fungus affects main crops and has led to great economic loss. Elicitors are a group of compounds that inspire the host plant to produce an immune response against invading pathogens. This study describes a protein that has high homology with the Trichoderma elicitor Sm1 and was found in the genome of S. sclerotiorum. We named this protein SsSm1. To determine whether this protein has an elicitor function like its homology protein, we constructed a heterologous expression vector for SsSm1 and expressed it in Escherichia coli. The protein of heterologous expression led to the formation of lesions in tobacco that closely resemble hypersensitive response lesions. Transient expression of the encoding gene of SsSm1 in tobacco leaves also caused hypersensitive response. Then, RNA silencing was used to identify the function of SsSm1. The hyphal growth and pathogenicity of silenced transformants were shown to be obviously lagging and branched abnormally. Transformants produced less infection cushions and deformed sclerotiorum. In addition, SsSm1 silencing caused weak tolerance to NaCl, sorbitol and SDS, and the sensitivity of mutants to carbendazim was also significantly decreased. Based on the above results, we speculate that this protein may be related to the development of hyphae, infection cushions and sclerotiorum, but the specific molecular mechanism needs to be studied further.