Characterization of necrosis-inducing NLP proteins in Phytophthora capsici

Abstract

BackgroundEffector proteins function not only as toxins to induce plant cell death, but also enable pathogens to suppress or evade plant defense responses. NLP-like proteins are considered to be effector proteins, and they have been isolated from bacteria, fungi, and oomycete plant pathogens. There is increasing evidence that NLPs have the ability to induce cell death and ethylene accumulation in plants.ResultsWe evaluated the expression patterns of 11 targeted PcNLP genes by qRT-PCR at different time points after infection by P. capsici. Several PcNLP genes were strongly expressed at the early stages in the infection process, but the expression of other PcNLP genes gradually increased to a maximum at late stages of infection. The genes PcNLP2, PcNLP6 and PcNLP14 showed the highest expression levels during infection by P. capsici. The necrosis-inducing activity of all targeted PcNLP genes was evaluated using heterologous expression by PVX agroinfection of Capsicum annuum and Nicotiana benthamiana and by Western blot analysis. The members of the PcNLP family can induce chlorosis or necrosis during infection of pepper and tobacco leaves, but the chlorotic or necrotic response caused by PcNLP genes was stronger in pepper leaves than in tobacco leaves. Moreover, PcNLP2, PcNLP6, and PcNLP14 caused the largest chlorotic or necrotic areas in both host plants, indicating that these three genes contribute to strong virulence during infection by P. capsici. This was confirmed through functional evaluation of their silenced transformants. In addition, we further verified that four conserved residues are putatively active sites in PcNLP1 by site-directed mutagenesis.ConclusionsEach targeted PcNLP gene affects cells or tissues differently depending upon the stage of infection. Most PcNLP genes could trigger necrotic or chlorotic responses when expressed in the host C. annuum and the non-host N. benthamiana. Individual PcNLP genes have different phytotoxic effects, and PcNLP2, PcNLP6, and PcNLP14 may play important roles in symptom development and may be crucial for virulence, necrosis-inducing activity, or cell death during infection by P. capsici.

Phytophthora capsici homologue of the cell cycle regulator SDA1 is required for sporangial morphology, mycelial growth and plant infection

“…The PCRs and parameters were adjusted slightly from those of Feng et al . (). PCRs were as follows: 94 °C for 3 min; followed by 40 cycles at 95 °C for 60 s, 55 °C for 35 s and 72 °C for 1 min; and a final extension at 72 °C for 10 min.…”

“…We used the plasmid vectors pHam 34 and pHspNpt (Feng et al ., ) to construct transformation plasmids. DNA fragments for the generation of constructs were amplified from DNA of P. capsici SD33 and digested with the restriction enzyme Sma I for cloning into the vector pHam 34.…”

“…A susceptible pepper cultivar (Capsicum annuum inbred line 06221) was used to evaluate the virulence of different strains. The seedlings of pepper line 06221 were cultivated as described previously (Feng et al, 2014). Briefly, single seedlings at the three-leaf stage were transplanted into small plastic trays and grown for 14 days before leaves were used in inoculation assays.…”

“…To measure the transcript levels of PcSDA1 during the early phases of P. capsici infection, the pepper leaf was inoculated with zoospores of the SD33 strain. Each leaflet was spot incubated with 2.0 μL of zoospore suspension (1 × 10 5 zoospores/mL) of the relevant strains and then kept in the dark at 25°C (Feng et al, 2014).…”

Molecular identification and characterization of Elsinoë murrayae (Synonym: Sphaceloma murrayae) from weeping willow

“…Most hemibiotrophs and necrotrophs whose NLP were tested, such as Fusarium oxysporum (Li & Durbin, 2009), Colletotrichum higginsianum (Kleemann et al, 2012), Mycosphaerella graminicola (Motteram et al, 2009), Botrytis cinerea (Schouten, van Baarlen, & van Kan, 2008) and Verticillium dahliae (Santhanam et al, 2013), have at least one cytotoxic NLP. Furthermore, cytotoxic NLP genes are usually highly expressed especially towards entering of the necrotrophic stage or during the necrotrophic stage (Cheng et al, 2014;Feng et al, 2014;Kleemann et al, 2012;Motteram et al, 2009;Qutob, Kamoun, & Gijzen, 2002). Thus, the cytotoxicity and expression patterns of widely distributed NLPs could serve as additional characteristics of fungal and fungal-like pathogens.…”