The rice TAL effector-dependent resistance protein XA10 triggers cell death and calcium depletion in the endoplasmic reticulum.

The recognition between disease resistance (R) genes in plants and their cognate avirulence (Avr) genes in pathogens can produce a hypersensitive response of localized programmed cell death. However, our knowledge of the early signaling events of the R gene-mediated hypersensitive response in plants remains limited. Here, we report the cloning and characterization of Xa10, a transcription activator-like (TAL) effector-dependent R gene for resistance to bacterial blight in rice (Oryza sativa). Xa10 contains a binding element for the TAL effector AvrXa10 (EBEAvrXa10) in its promoter, and AvrXa10 specifically induces Xa10 expression. Expression of Xa10 induces programmed cell death in rice, Nicotiana benthamiana, and mammalian HeLa cells. The Xa10 gene product XA10 localizes as hexamers in the endoplasmic reticulum (ER) and is associated with ER Ca(2+) depletion in plant and HeLa cells. XA10 variants that abolish programmed cell death and ER Ca(2+) depletion in N. benthamiana and HeLa cells also abolish disease resistance in rice. We propose that XA10 is an inducible, intrinsic terminator protein that triggers programmed cell death by a conserved mechanism involving disruption of the ER and cellular Ca(2+) homeostasis.

Genetic engineering of the Xa10 promoter for broad-spectrum and durable resistance to Xanthomonas oryzae pv. oryzae.

Many pathovars of plant pathogenic bacteria Xanthomonas species inject transcription activator-like (TAL) effectors into plant host cells to promote disease susceptibility or trigger disease resistance. The rice TAL effector-dependent disease resistance gene Xa10 confers narrow-spectrum race-specific resistance to Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight disease in rice. To generate broad-spectrum and durable resistance to Xoo, we developed a modified Xa10 gene, designated as Xa10(E5) . Xa10(E5) has an EBE-amended promoter containing 5 tandemly arranged EBEs each responding specifically to a corresponding virulent or avirulent TAL effector and a stable transgenic rice line containing Xa10(E5) was generated in the cultivar Nipponbare. The Xa10(E5) gene was specifically induced by Xoo strains that harbour the corresponding TAL effectors and conferred TAL effector-dependent resistance to the pathogens at all developmental stages of rice. Further disease evaluation demonstrated that the Xa10(E5) gene in either Nipponbare or 9311 genetic backgrounds provided broad-spectrum disease resistance to 27 of the 28 Xoo strains collected from 11 countries. The development of Xa10(E5) and transgenic rice lines provides new genetic materials for molecular breeding of rice for broad-spectrum and durable disease resistance to bacterial blight.

Inactivation of retrotransposon Tos17 Chr.7 in rice cultivar Nipponbare through CRISPR/Cas9-mediated gene editing.

Retrotransposons are mobile genetic elements capable of transposition via reverse transcription of RNA intermediates. Rice cultivar Nipponbare contains two nearly identical genomic copies of Tos17, an endogenous copia-like LTR retrotransposon, on chromosomes 7 (Tos17 Chr.7) and 10 (Tos17 Chr.10), respectively. Previous studies demonstrated that only Tos17 Chr.7 is active in transposition during tissue culture. Tos17 Chr.7 has been extensively used for insertional mutagenesis as a tool for functional analysis of rice genes. However, Tos17 Chr.7 transposition might generate somaclonal mutagenesis with undesirable traits during rice transformation, which would affect the evaluation or application of transgenes. In this study, we generated a Tos17 Chr.7 knockout mutant D873 by using CRISPR/Cas9 gene editing system. The gene-edited allele of Tos17 Chr.7 in D873, designated as Tos17 D873, has an 873-bp DNA deletion in the pol gene of Tos17 Chr.7, which caused the deletion of the GAG-pre-integrase domain and the integrase core domain. Although the transcription of Tos17 D873 was activated in D873 calli, no transposition of Tos17 D873 was detected in the regenerated D873 plants. The results demonstrate that the GAG-pre-integrase domain and the integrase core domain are essential for Tos17 Chr.7 transposition and the deletion of the two domains could be not complemented by other LTR retrotransposons in rice genome. As the Tos17 Chr.7-derived somaclonal mutagenesis is blocked in the D873 plants, the generation of the Tos17 D873 allele will be helpful in production of transgenic rice plants for gene function study and genetic engineering. Similar approach can be used to inactivate other retrotransposons in crop breeding.

Marker-assisted breeding of the rice restorer line Wanhui 6725 for disease resistance, submergence tolerance and aromatic fragrance.

BACKGROUND: Rice is a staple food crop in the world. With the increase in world population and economic development, farmers need to produce more rice in limited field. However, the rice production is frequently affected by biotic and abiotic stresses. The use of natural disease resistance and stress tolerance through genetic breeding is the most efficient and economical way to combat or acclimate to these stresses. In addition, rice with aromatic fragrance can significantly increase market value for its good grain quality. Mianhui 725 (MH725) is an elite restorer line that has been widely used to produce three-line hybrid rice in China. We previously introduced rice bacterial blight resistance genes Xa4 and Xa21 into MH725 and obtained an introgression rice line Wanhui 421 (WH421), which theoretically possesses 96.9% genetic background of MH725. RESULTS: Here we report the introduction and pyramiding of disease resistance genes Xa27 and Pi9, submergence tolerance gene Sub1A and aromatic fragrance gene badh2.1 in WH421 through backcrossing and marker-assisted selection. The newly developed introgression rice line was designated as Wanhui 6725 (WH6725), which theoretically possesses 95.0% genetic background of MH725. WH6725 and its hybrid rice conferred disease resistance to both blast and bacterial blight diseases and showed tolerance to submergence for over 14 days without significant loss of viability. Compared with non-aromatic rice MH725, WH6725 has strong aromatic fragrance. The major important agronomic traits and grain quality of WH6725 and its hybrid rice obtained in field trials were similar to those of MH725 and the control hybrid rice, indicating that WH6725 is as good as MH725 when it is used as a restorer line for three-line hybrid rice production. CONCLUSION: We have successfully developed a new restorer line WH6725 with disease resistance to rice blast and bacterial blight, tolerance to submergence and aromatic fragrance, which can be used to replace MH725 for hybrid rice production.

[Inheritance and expression of the maize pepc gene in progenies of transgenic rice bred by crossing].

By cross breeding, the maize pepc gene in the pepc transgenic rice was successfully incorporated into the parents of two-, three-line hybrid rice, including sterile lines (Peiai64S, 2302S, 2304S, 2306S and Shuangjiu A) and restorer lines (5129, 02428 and Wanjing97) to breed the high-photosynthetic efficiency parents of hybrid rice and utilize heterosis between C4 and C4/C3 rice. Some lines of pepc transgenic rice (LPTR) have been developed. The study on the generations of LPTR suggests the following: (1) The segregation observed in F2 and BC1 progenies demonstrated that pepc transgene inherited as a single dominant gene in the progenies of LPTR. (2) The maize pepc gene is actively expressed at high level in LPTR, and changes of pepc gene expression in the progenies of LPTR may be related to position effect, difference of gene copy number and environmental factors. (3) Through the selection method of soaking seeds into hygromycin solution to germinate, tracing the pepc gene by PCR analysis, evaluating the performance of the rice plants in the field and examining PEPC activities, the segregation of the pepc transgene in LPTR was controlled effectively. Based on the above strategy three pepc transgene lines, H1596, H1597 and Y1470, have been selected. The result suggests that it is possible to breed practical, stable and high-expression pepc transgenic rice by conventional crossing.

Marker-assisted breeding of Indonesia local rice variety Siputeh for semi-dwarf phonetype, good grain quality and disease resistance to bacterial blight.

BACKGROUND: Rice is one of the most important staple food crops in Asia. Since the first green revolution beginning in 1960s, high-yield semidwarf modern rice varieties have been widely planted; however, traditional rice varieties with tall plant type are still grown in many countries due to their good grain quality and adaptation to local climate and environment. Siputeh, a local rice variety mainly planted in Java and Sumatra islands of Indonesia, produces long grain rice with good cooking and eating quality. However, the variety has low yield with tall plant type and long growth duration and is highly susceptible to biotic and abiotic stress. RESULTS: Siputeh as the recurrent female was crossed with the donor line WH421, an elite paternal line of hybrid rice containing the sd1, Wx (b), Xa4 and Xa21 genes, followed by backcrossing and self-pollination. TS4, a BC3F4 line derived from the breeding program, was obtained through marker-assisted selection for the sd1, Wx (b), Xa4 and Xa21 loci. TS4 has semi-dwarf phenotype and short growth duration. TS4 conferred disease resistance to multiple Xanthomonas oryzae pv. oryzae (Xoo) strains collected from different countries around the world. TS4 achieved higher grain yield than Siputeh in two field trials conducted in Banda Aceh, Indonesia and Lingshui, China, respectively. Finally, TS4 has better grain quality than Siputeh in terms of degree of chalkiness and amylose content. CONCLUSION: An improved rice line, designed as TS4, has been developed to contain semi-dwarf gene sd1, low amylase content gene Wx (b) and bacterial light resistance genes Xa4 and Xa21 through marker-assisted selection. TS4 has semi-dwarf phenotype with reduced growth duration, produces high yield with good grain quality and provides broad-spectrum resistance to Xoo strains. The development of TS4 enriches the diversity of local rice varieties with high yield potential and good grain quality.