QQS orphan gene and its interactor NF-YC4 reduce susceptibility to pathogens and pests

Abstract

Enhancing the nutritional quality and disease resistance of crops without sacrificing productivity is a key issue for developing varieties that are valuable to farmers and for simultaneously improving food security and sustainability. Expression of the Arabidopsis thaliana species-specific AtQQS (Qua-Quine Starch) orphan gene or its interactor, NF-YC4 (Nuclear Factor Y, subunit C4), has been shown to increase levels of leaf/seed protein without affecting the growth and yield of agronomic species. Here, we demonstrate that overexpression of AtQQS and NF-YC4 in Arabidopsis and soybean enhances resistance/reduces susceptibility to viruses, bacteria, fungi, aphids and soybean cyst nematodes. A series of Arabidopsis mutants in starch metabolism were used to explore the relationships between QQS expression, carbon and nitrogen partitioning, and defense. The enhanced basal defenses mediated by QQS were independent of changes in protein/carbohydrate composition of the plants. We demonstrate that either AtQQS or NF-YC4 overexpression in Arabidopsis and in soybean reduces susceptibility of these plants to pathogens/pests. Transgenic soybean lines overexpressing NF-YC4 produce seeds with increased protein while maintaining healthy growth. Pull-down studies reveal that QQS interacts with human NF-YC, as well as with Arabidopsis NF-YC4, and indicate two QQS binding sites near the NF-YC-histone-binding domain. A new model for QQS interaction with NF-YC is speculated. Our findings illustrate the potential of QQS and NF-YC4 to increase protein and improve defensive traits in crops, overcoming the normal growth-defense trade-offs.

Keywords: QQS; NF-YC4; carbon and nitrogen partitioning; orphan; pathogen; pest.

Figure 1

Arabidopsis mutants of QQS and its interactor NF‐YC4 have altered susceptibility to viral and bacterial infection. Mutants: transgenic AtQQS RNAi, AtQQS‐OE and AtNF‐YC4‐OE in Arabidopsis Col‐0* (few trichomes), and T‐DNA knockout mutants Atqqs, and Atnf‐yc4 in Col‐0 (trichomes). (a) The numbers of TuMV‐GFP infection foci at 120 HAI, (b) the sizes of TuMV‐GFP infection foci at 120 HAI and (c) the growth of the Pst DC3000 (DC3000) and CUCPB5115 (Pst DC3000 ΔCEL) bacterial strains were altered in the mutants. CFU, colony forming units. All data in bar charts show mean ± SE (standard error), n = 3. Statistical significance was determined as described in Appendix S1 ‘Experiment design and statistical methods’: ***P < 0.001; **P < 0.01; *P < 0.05; •P < 0.1.

Figure 2

Transgenic soybean lines constitutively expressing AtQQS or overexpressing GmNF‐YC4‐1 had decreased viral and bacterial infection. (a) Systemic infection of BPMV was decreased at both 11 and 13 DPI. (b) Growth of PsgR4 was decreased at 7 DPI. CFU, colony forming units. All data in bar charts show mean ± SE, n = 3. ***P < 0.001; **P < 0.01; *P < 0.05; •P < 0.1.

Figure 3

Aphid, SCN and SDS performance were altered in soybean AtQQS‐E and GmNF‐YC4‐1‐OE mutants. (a) Aphid number, (b) number of soybean cyst nematode females and (c) foliar disease index were decreased in mutants. FI, female index; Williams 82 and Jack lines of soybean, controls for highly susceptible and resistant, respectively. Bar graphs show mean ± SE, n = 10 (a) or 6 (b and c). ***P < 0.001; **P < 0.01; *P < 0.05; •P < 0.1.

Figure 4

Virus infection of Arabidopsis starch mutants is correlated with QQS transcript level. (a) Leaf starch accumulation, (b) the transcript levels of QQS and NF‐YC4 in mutants, quantified by real‐time PCR, and (c) leaf protein content, at the end of light period. (d) The average sizes of TuMV‐GFP infection foci at 120 HAI were increased in plants with down‐regulated AtQQS and decreased in plants overexpressing AtQQS . Bar charts show mean ± SE, n = 3. ***P < 0.001; **P < 0.01; *P < 0.05.

Figure 5

QQS binding with NF‐YC4. (a) QQS fragments used for pull‐down assays. NF‐YC fragments used are shown in Figure S5. (b) MBP pull‐down assays: QQS binds to AtNF‐YC4 in the regions of aa 1‐12 and aa 41‐59. (c) MBP pull‐down assays: QQS binds to HsNF‐YC in the regions of aa 1‐12 and aa 41‐59. (d) GST pull‐down assays: QQS fragments bind to AtNF‐YC4, full‐length HsNF‐YC and the first‐145‐aa N‐terminal of HsNF‐YC. Blue font, no interaction, red font, interaction between QQS fragments and NF‐YC.

Figure 6

QQS binding sites with NF‐YC and speculated model for QQS and NF‐YC4‐induced changes in composition and plant defense. (a) Model of the interactions of NF‐YB‐51‐57, NF‐YC, and NF‐YA, and the QQS‐5‐11 interactions with NF‐YC. (b) Model of the interactions of NF‐YB‐62‐70 and NF‐YC, and the QQS‐41‐49 interactions with NF‐YC. NF‐YB‐62‐70 are not in contact with NF‐YA, so NF‐YA is not represented here. See Figure S6a,b for detailed explanation of the models, which are based on analysis of previously published crystal structure data [Protein Database Bank (PDB) IDs: 1N1J (Romier et al., 2003) and 4 AWL (Nardini et al., 2013)]. (c) QQS binds to two NF‐YC hydrophobic interfaces. Based on the sequence similarity between QQS‐5‐11 and QQS‐41‐49 and the NF‐YB N‐terminal region, we propose QQS binds to two NF‐YC hydrophobic interfaces that the NF‐YB N‐terminal region binds to (Figure 6a,b). However, NF‐YB only has three residues (aa from 58 to 61, solid light blue line) in α1 to link the two binding sites and QQS has a 29‐residue long fragment (aa 12–40, dashed yellow line) to link the QQS N‐ and C‐terminal binding sites. Shape of the residues: octagon, NF‐YA; ellipse, NF‐YB; circle, NF‐NC; and shaded rectangle, QQS. Colour of residues represents the polarity and hydrophobicity: red, aliphatic; purple, negatively charged; blue, positively charged; light blue, polar; green, aromatic; and yellow, unique Pro (P). The distance cut‐offs for interaction: 5 Å for hydrophobic interaction, 6 Å for ionic interaction and cation‐pi interaction, and 3.5 Å and 4.0 Å for hydrogen bond when the donor is oxygen/nitrogen and sulphur, respectively. Solid line represents the interaction in both 1N1J and 4 AWL, the broken line in 1N1J only, and the dashed line in 4 AWL only. The colour of the lines represents the type of interaction: dark red, hydrophobic interaction; light blue, hydrogen bond; dark blue, ionic interaction; and black, cation‐pi interaction. The residue marked with * is in contact with DNA within 5 Å. (d) Proposed model of defense priming by QQS: QQS/NF‐YC4 protein complex moves to the nucleus, binding NF‐YA to regulate transcription of downstream genes. QQS may compete with NF‐YB to bind NF‐YC, thus altering the NF‐Y protein complex.