Host‐induced gene silencing of an important pathogenicity factor PsCPK1 in Puccinia striiformis f. sp. tritici enhances resistance of wheat to stripe rust

Rust fungi are devastating plant pathogens and cause a large economic impact on wheat production worldwide. To overcome this rapid loss of resistance in varieties, we generated stable transgenic wheat plants expressing short interfering RNAs (siRNAs) targeting potentially vital genes of Puccinia striiformis f. sp. tritici (Pst). Protein kinase A (PKA) has been proved to play important roles in regulating the virulence of phytopathogenic fungi. PsCPK1, a PKA catalytic subunit gene from Pst, is highly induced at the early infection stage of Pst. The instantaneous silencing of PsCPK1 by barley stripe mosaic virus (BSMV)‐mediated host‐induced gene silencing (HIGS) results in a significant reduction in the length of infection hyphae and disease phenotype. These results indicate that PsCPK1 is an important pathogenicity factor by regulating Pst growth and development. Two transgenic lines expressing the RNA interference (RNAi) construct in a normally susceptible wheat cultivar displayed high levels of stable and consistent resistance to Pst throughout the T₃ to T₄ generations. The presence of the interfering RNAs in transgenic wheat plants was confirmed by northern blotting, and these RNAs were found to efficiently down‐regulate PsCPK1 expression in wheat. This study addresses important aspects for the development of fungal‐derived resistance through the expression of silencing constructs in host plants as a powerful strategy to control cereal rust diseases.     

Expression of apoplast-targeted plant defensin <Emphasis Type="Italic">MtDef4.2</Emphasis> confers resistance to leaf rust pathogen <Emphasis Type="Italic">Puccinia triticina</Emphasis> but does not affect mycorrhizal symbiosis in transgenic wheat

Rust fungi of the order Pucciniales are destructive pathogens of wheat worldwide. Leaf rust caused by the obligate, biotrophic basidiomycete fungus Puccinia triticina (Pt) is an economically important disease capable of causing up to 50 % yield losses. Historically, resistant wheat cultivars have been used to control leaf rust, but genetic resistance is ephemeral and breaks down with the emergence of new virulent Pt races. There is a need to develop alternative measures for control of leaf rust in wheat. Development of transgenic wheat expressing an antifungal defensin offers a promising approach to complement the endogenous resistance genes within the wheat germplasm for durable resistance to Pt. To that end, two different wheat genotypes, Bobwhite and Xin Chun 9 were transformed with a chimeric gene encoding an apoplast-targeted antifungal plant defensin MtDEF4.2 from Medicago truncatula. Transgenic lines from four independent events were further characterized. Homozygous transgenic wheat lines expressing MtDEF4.2 displayed resistance to Pt race MCPSS relative to the non-transgenic controls in growth chamber bioassays. Histopathological analysis suggested the presence of both pre- and posthaustorial resistance to leaf rust in these transgenic lines. MtDEF4.2 did not, however, affect the root colonization of a beneficial arbuscular mycorrhizal fungus Rhizophagus irregularis. This study demonstrates that the expression of apoplast-targeted plant defensin MtDEF4.2 can provide substantial resistance to an economically important leaf rust disease in transgenic wheat without negatively impacting its symbiotic relationship with the beneficial mycorrhizal fungus.     

The Lr34 adult plant rust resistance gene provides seedling resistance in durum wheat without senescence

The hexaploid wheat (Triticum aestivum) adult plant resistance gene, Lr34/Yr18/Sr57/Pm38/Ltn1, provides broad‐spectrum resistance to wheat leaf rust (Lr34), stripe rust (Yr18), stem rust (Sr57) and powdery mildew (Pm38) pathogens, and has remained effective in wheat crops for many decades. The partial resistance provided by this gene is only apparent in adult plants and not effective in field‐grown seedlings. Lr34 also causes leaf tip necrosis (Ltn1) in mature adult plant leaves when grown under field conditions. This D genome‐encoded bread wheat gene was transferred to tetraploid durum wheat (T. turgidum) cultivar Stewart by transformation. Transgenic durum lines were produced with elevated gene expression levels when compared with the endogenous hexaploid gene. Unlike nontransgenic hexaploid and durum control lines, these transgenic plants showed robust seedling resistance to pathogens causing wheat leaf rust, stripe rust and powdery mildew disease. The effectiveness of seedling resistance against each pathogen correlated with the level of transgene expression. No evidence of accelerated leaf necrosis or up‐regulation of senescence gene markers was apparent in these seedlings, suggesting senescence is not required for Lr34 resistance, although leaf tip necrosis occurred in mature plant flag leaves. Several abiotic stress‐response genes were up‐regulated in these seedlings in the absence of rust infection as previously observed in adult plant flag leaves of hexaploid wheat. Increasing day length significantly increased Lr34 seedling resistance. These data demonstrate that expression of a highly durable, broad‐spectrum adult plant resistance gene can be modified to provide seedling resistance in durum wheat.     

Interspecies gene transfer provides soybean resistance to a fungal pathogen

Fungal pathogens pose a major challenge to global crop production. Crop varieties that resist disease present the best defence and offer an alternative to chemical fungicides. Exploiting durable nonhost resistance (NHR) for crop protection often requires identification and transfer of NHR‐linked genes to the target crop. Here, we identify genes associated with NHR of Arabidopsis thaliana to Phakopsora pachyrhizi, the causative agent of the devastating fungal disease called Asian soybean rust. We transfer selected Arabidopsis NHR‐linked genes to the soybean host and discover enhanced resistance to rust disease in some transgenic soybean lines in the greenhouse. Interspecies NHR gene transfer thus presents a promising strategy for genetically engineered control of crop diseases.     

Host‐induced gene silencing of an essential chitin synthase gene confers durable resistance to Fusarium head blight and seedling blight in wheat

Fusarium head blight (FHB) and Fusarium seedling blight (FSB) of wheat, caused by Fusarium pathogens, are devastating diseases worldwide. We report the expression of RNA interference (RNAi) sequences derived from an essential Fusarium graminearum (Fg) virulence gene, chitin synthase (Chs) 3b, as a method to enhance resistance of wheat plants to fungal pathogens. Deletion of Chs3b was lethal to Fg; disruption of the other Chs gene family members generated knockout mutants with diverse impacts on Fg. Comparative expression analyses revealed that among the Chs gene family members, Chs3b had the highest expression levels during Fg colonization of wheat. Three hairpin RNAi constructs corresponding to the different regions of Chs3b were found to silence Chs3b in transgenic Fg strains. Co‐expression of these three RNAi constructs in two independent elite wheat cultivar transgenic lines conferred high levels of stable, consistent resistance (combined type I and II resistance) to both FHB and FSB throughout the T₃ to T₅ generations. Confocal microscopy revealed profoundly restricted mycelia in Fg‐infected transgenic wheat plants. Presence of the three specific short interfering RNAs in transgenic wheat plants was confirmed by Northern blotting, and these RNAs efficiently down‐regulated Chs3b in the colonizing Fusarium pathogens on wheat seedlings and spikes. Our results demonstrate that host‐induced gene silencing of an essential fungal chitin synthase gene is an effective strategy for enhancing resistance in crop plants under field test conditions.     

The wheat durable,multipathogen resistance gene Lr34 confers partial blast resistance in rice

The wheat gene Lr34 confers durable and partial field resistance against the obligate biotrophic, pathogenic rust fungi and powdery mildew in adult wheat plants. The resistant Lr34 allele evolved after wheat domestication through two gain‐of‐function mutations in an ATP‐binding cassette transporter gene. An Lr34‐like fungal disease resistance with a similar broad‐spectrum specificity and durability has not been described in other cereals. Here, we transformed the resistant Lr34 allele into the japonica rice cultivar Nipponbare. Transgenic rice plants expressing Lr34 showed increased resistance against multiple isolates of the hemibiotrophic pathogen Magnaporthe oryzae, the causal agent of rice blast disease. Host cell invasion during the biotrophic growth phase of rice blast was delayed in Lr34‐expressing rice plants, resulting in smaller necrotic lesions on leaves. Lines with Lr34 also developed a typical, senescence‐based leaf tip necrosis (LTN) phenotype. Development of LTN during early seedling growth had a negative impact on formation of axillary shoots and spikelets in some transgenic lines. One transgenic line developed LTN only at adult plant stage which was correlated with lower Lr34 expression levels at seedling stage. This line showed normal tiller formation and more importantly, disease resistance in this particular line was not compromised. Interestingly, Lr34 in rice is effective against a hemibiotrophic pathogen with a lifestyle and infection strategy that is different from obligate biotrophic rusts and mildew fungi. Lr34 might therefore be used as a source in rice breeding to improve broad‐spectrum disease resistance against the most devastating fungal disease of rice.     

TECHNICAL ADVANCE: Transformation of the flax rust fungus,Melampsora lini: selection via silencing of an avirulence gene

Rust fungi cause devastating diseases on many important food crops, with a damaging stem rust epidemic currently affecting wheat production in Africa and the Middle East. These parasitic fungi propagate exclusively on plants, precluding the use of many biotechnological tools available for other culturable fungi. In particular the lack of a stable transformation system has been an impediment to the genetic manipulation required for molecular analysis of rust pathogenicity. We have developed an Agrobacterium‐mediated genetic transformation procedure for the model flax rust fungus Melampsora lini, which infects flax (Linum usitatissimum). Selection of transgenic rust lines is based on silencing of AvrL567, which encodes a rust effector protein that is recognised by the flax L6 immune receptor. The non‐transgenic rust line is unable to infect flax plants expressing L6, while silenced transgenic lines are virulent on these plants, providing an effective selection system. This directly confirms that the cloned AvrL567 gene is responsible for flax rust virulence phenotypes, and demonstrates the utility of this system to probe rust gene function.     

Effect of Host Plant Resistance on Haustorium Formation in Cereal Rust Fungi

Haustoria of Puccinia triticina (wheat leaf rust fungus) and P. hordei (barley leaf rust fungus) were isolated from susceptible and partially resistant wheat lines, and susceptible, hypersensitive and partially resistant barley lines. Haustoria were counted and measured. The size of haustoria was similar in the partially resistant and susceptible genotypes but haustoria were smaller in the hypersensitive barley line L94+Pa7. The number of haustoria was reduced in both partially and hypersensitive lines when compared with susceptible ones. Therefore it seems that the reduction in the number of haustoria is a consequence of the resistance that can be attributable either to early abortion of infection units or reduced colony growth. The reduction of the number of haustoria was more pronounced in the adult plant stage.     

另辟蹊径破解小麦条锈病的基因密码

小麦条锈病是由条形柄锈菌小麦专化型(Puccinia striiformis f. sp. tritici, Pst)引起的真菌病害, 在全世界范围内危害小麦(Triticum aestivum)生产。培育和种植持久抗性小麦品种是控制小麦条锈病最有效的方法。由于病原体突变导致免疫受体逃避检测, 因此抗病基因经常失效。而易感基因(S基因)突变介导的抗性常具持久性与广谱性。近日, 西北农林科技大学植物免疫研究团队在揭示小麦受S基因保护的分子机制方面取得显著进展, 为抗病育种提供了有力工具。他们发现小麦感染条锈菌后, 真菌诱导受体样细胞质激酶TaPsIPK1与效应子PsSpg1特异性互作, 通过增强激酶活性和TaPsIPK1进入细胞核促进寄生。TaPsIPK1磷酸化转录因子TaCBF1d。TaCBF1d的磷酸化改变了其下游基因的转录活性。因此, TaPsIPK1和PsSpg1增强TaCBF1d磷酸化可能会重新编程靶基因表达, 干扰植物防御反应, 从而促进病原体感染。在2年的田间试验中, 小麦中TaPsIPK1的CRISPR-Cas9失活赋予了对Pst的广谱抗性, 且不影响重要的农艺性状。该研究首次揭示了由PsSpg1-TaPsIPK1-TaCBF1d在小麦条锈病S基因中触发的新的磷酸化转录调控机制, 为通过作物遗传修饰培育持久抗性品种提供了新策略。     

Phenotypic and genetic diversity of leaf rust resistance in wheat wild relatives

Puccinia triticina (Pt), the causal agent of leaf rust evolves through forming new pathotypes that adversely affect the growth and yield of wheat cultivars. Therefore, continued production of resistant varieties through exploring novel sources of resistance in wild relatives which are abundantly found in Iran and the neighbouring regions is a major task in wheat breeding programs. The aim of the present study was to explore 60 wild wheat genotypes selected from the species Triticum monococcum, Aegilops tauschii, Ae. neglecta, Ae. cylindrica, Ae. triuncialis, Ae. umbellulata, Ae. speltoides, Ae. columnaris, Ae. crassa and Ae. ventricosa for resistance to leaf rust. The cultivar ‘Boolani’ and Thatcher near-isogenic lines were used as controls. Two-week-old seedlings were inoculated using 10 Pt pathotypes, and the infection types were recorded. The genotypes were also analysed for polymorphism using six sequence-tagged sites (STS) and sequence characterized amplified region (SCAR) markers. Forty-eight genotypes produced high infection types (3⁺) for two pathotypes, but the remaining genotypes produced low infection types of ‘0; ^=’ to ‘1⁺CN’ to all pathotypes. The latter included three accessions of Ae. tauschii, two accessions of each Ae. umbellulata, Ae. columnaris and Triticum monococcum, and one accession from each Ae. triuncialis, Ae. ventricosa and Ae. neglecta. Analysis for STS and SCAR markers suggested several genotypes could carry the genes Lr9, Lr10, Lr19, Lr24, Lr26 and Lr37 or their potential orthologs in addition to unknown resistance genes. In conclusion, the identified resistant genotypes could be further characterized and used in wheat breeding programs for leaf rust resistance.     

Molecular Detection of Stripe Rust Resistance Gene(s) in 115 Wheat Cultivars (lines) from the Yellow and Huai River Valley Wheat Region

Stripe rust (yellow rust), caused by Puccinia striiformis f.sp. tritici (Pst), is a serious disease of wheat worldwide, including China. Growing resistant cultivars is the most cost‐effective and environmentally friendly approach to control the disease. To assess the stripe rust resistance in commercial wheat cultivars and advanced lines in the Yellow and Huai River Valley Wheat Region, 115 wheat cultivars (lines) collected from 13 provinces in this region were evaluated with the most prevalent Chinese Pst races CYR32, CYR33 and the new race V26 at seedling stage. In addition, these wheat entries were inoculated with the mixed races of CYR32 and CYR33 at the adult‐plant stage in the field. The results indicated that 53 (46.1%) cultivars (lines) had all‐stage resistance to all the three races, and 16 (13.9%) cultivars (lines) showed adult‐plant resistance. The possible stripe rust resistance genes in these entries were postulated by the closely linked markers of all‐stage resistance genes Yr5, Yr9, Yr10, Yr15 and Yr26 and adult‐plant resistance gene Yr18. Molecular analysis indicated that resistance genes Yr5, Yr9, Yr10, Yr18 and Yr26 were found in 5 (4.3%), 38 (33.0%), 1 (0.9%), 2 (1.7%) and 8 (7.0%) entries, respectively. No entry was found to carry the Yr15 gene. In future breeding programs, Yr5, Yr15 and Yr18 should be used to pyramid with other effective genes to develop wheat cultivars with high‐level and durable resistance to stripe rust, whereas Yr9, Yr10 and Yr26 should not be used or used in a limited way due to the virulent races present in China.     

Next‐generation transgenic cotton: pyramiding RNAi and Bt counters insect resistance

Transgenic crops producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) are extensively cultivated worldwide. To counter rapidly increasing pest resistance to crops that produce single Bt toxins, transgenic plant ‘pyramids’ producing two or more Bt toxins that kill the same pest have been widely adopted. However, cross‐resistance and antagonism between Bt toxins limit the sustainability of this approach. Here we describe development and testing of the first pyramids of cotton combining protection from a Bt toxin and RNA interference (RNAi). We developed two types of transgenic cotton plants producing double‐stranded RNA (dsRNA) from the global lepidopteran pest Helicoverpa armigera designed to interfere with its metabolism of juvenile hormone (JH). We focused on suppression of JH acid methyltransferase (JHAMT), which is crucial for JH synthesis, and JH‐binding protein (JHBP), which transports JH to organs. In 2015 and 2016, we tested larvae from a Bt‐resistant strain and a related susceptible strain of H. armigera on seven types of cotton: two controls, Bt cotton, two types of RNAi cotton (targeting JHAMT or JHBP) and two pyramids (Bt cotton plus each type of RNAi). Both types of RNAi cotton were effective against Bt‐resistant insects. Bt cotton and RNAi acted independently against the susceptible strain. In computer simulations of conditions in northern China, where millions of farmers grow Bt cotton as well as abundant non‐transgenic host plants of H. armigera, pyramided cotton combining a Bt toxin and RNAi substantially delayed resistance relative to using Bt cotton alone.     

Backcross-breeding and doubled-haploid facilitated introgression of stripe rust resistance in bread wheat

Stripe rust caused by the fungus Puccinia striiformis f. sp. tritici (Pst) may decrease wheat yield significantly in severe outbreaks. The most cost-effective and environmentally friendly approach to reduce yield losses due to rust diseases is deployment of effective resistant genes in wheat cultivars. The causal agents evolve and may break existing resistant sources as well. Therefore, long-term conventional breeding strategies and the ongoing evolution of pathogen populations in the region would put the success of breeding programmes at risk so that there is always a need for speeding up the process of germplasm enhancement through production of doubled-haploid breeding materials. In this study, we aimed at introgression of stripe rust resistance trait from three genotypes (Flanders, Martonvasar-17 (MV17) and Bersee) into a widely adapted cultivar “Ghods”. Positively selected F2BC² progenies of three backcrossing schemas, i.e. (i) Flanders/3^*Ghods; (ii) Ghods^*3/MV17; and (iii) Hybride-de-bersee/3^*Ghods, were used to produce three small-size doubled-haploid populations via wheat × Maize pollination methodology. The doubled-haploid populations were examined against two predominantly isolates of P. striiformis f. sp. tritici (Pst) i.e. 6E134A⁺ and 6E2A⁺Yr27⁺ and the screening revealed that 44 and 52 of the progenies are resistant to the above-mentioned isolates, respectively. Field data have shown that the stripe rust resistance doubled-haploid germplasm are comparable to local check cultivars in yield and earliness.     

Effective and ineffective resistance genes to wheat yellow rust during six years monitoring in Ardabil

Stripe (yellow) rust caused by Puccinia striiformis f. sp. tritici is the most devastating disease of bread wheat (Triticum aestivum) in the cool winter areas. This rust disease represents a constant threat to wheat production in several countries in Central and Western Asia. A wide range of virulent yellow rust pathotypes is evolving in this region causing the breakdown of widely utilised sources of resistance in wheat. Hence, the knowledge of effective resistance genes in the region will enable breeders to target those useful genes in their breeding programmes. From 2006 to 2012, in order to determine of effective resistance genes in Ardabil, north-west of Iran, virulence patterns of wheat yellow rust were studied under the field conditions by planting of differential sets and isogenic lines. The results showed that yellow rust resistance genes Yr1,Yr2⁺ , Yr3V, Yr3a, Yr4a, Yr4, Yr5, Yr7⁺ , Yr10, Yr15,Yr16, YrCV, YrSD and YrND were effective and race-nonspecific resistance genes YrA3, YrA4, Yr18 and Yr29 were partially effective during study periods. Genes Yr2, Yr6, Yr7, Yr9, Yr17, Yr20, Yr21,Yr22, Yr23, Yr24, Yr25, Yr26, Yr27, YrSU, YrSP and YrA were found ineffective. The Genes found effective against yellow rust under natural conditions may be deployed singly or in combinations with durable resistance genes to develop high yielding resistant wheat cultivars in wheat-growing areas in where yellow rust races have the same virulence profile to the prevalent race/s of Ardabil.