Two genomic regions of a sodium azide induced rice mutant confer broad-spectrum and durable resistance to blast disease

Rice blast, one of the most destructive epidemic diseases, annually causes severe losses in grain yield worldwide. To manage blast disease, breeding resistant varieties is considered a more economic and environment-friendly strategy than chemical control. For breeding new resistant varieties, natural germplasms with broad-spectrum resistance are valuable resistant donors, but the number is limited. Therefore, artificially induced mutants are an important resource for identifying new broad-spectrum resistant (R) genes/loci. To pursue this approach, we focused on a broad-spectrum blast resistant rice mutant line SA0169, which was previously selected from a sodium azide induced mutation pool of TNG67, an elite japonica variety. We found that SA0169 was completely resistant against the 187 recently collected blast isolates and displayed durable resistance for almost 20 years. Linkage mapping and QTL-seq analysis indicated that a 1.16-Mb region on chromosome 6 (Pi169-6(t)) and a 2.37-Mb region on chromosome 11 (Pi169-11(t)) conferred the blast resistance in SA0169. Sequence analysis and genomic editing study revealed 2 and 7 candidate R genes in Pi169-6(t) and Pi169-11(t), respectively. With the assistance of mapping results, six blast and bacterial blight double resistant lines, which carried Pi169-6(t) and/or Pi169-11(t), were established. The complementation of Pi169-6(t) and Pi169-11(t), like SA0169, showed complete resistance to all tested isolates, suggesting that the combined effects of these two genomic regions largely confer the broad-spectrum resistance of SA0169. The sodium azide induced mutant SA0169 showed broad-spectrum and durable blast resistance. The broad resistance spectrum of SA0169 is contributed by the combined effects of two R regions, Pi169-6(t) and Pi169-11(t). Our study increases the understanding of the genetic basis of the broad-spectrum blast resistance induced by sodium azide mutagenesis, and lays a foundation for breeding new rice varieties with durable resistance against the blast pathogen.

Simultaneous overexpression of multiple herbicide-metabolizing genes for broad-spectrum resistance in an agricultural weed Echinochloa phyllopogon

The use of herbicides in agricultural fields has driven the evolution of weeds for resistance, causing a grave threat to the current agriculture. One big mystery of weed resistance involves multiple-herbicide resistance (MHR) concomitant to enhanced herbicide metabolism. Previous research unveiled that the overexpression of catalytically promiscuous cytochrome P450s underlies the metabolism-based cross-resistance in multiple species. However, the concept of activation of promiscuous enzymes does not fully accommodate the resistance to diverse herbicides in MHR Echinochloa phyllopogon although the genetic inheritance of MHR was suggested as under a single gene control. Here, we show that the high-level resistance to diclofop-methyl in E. phyllopogon is caused by the simultaneous overexpression of CYP81A12/21, the previously identified promiscuous P450s, and a novel P450 CYP709C69. We found that the MHR line rapidly produced two distinct hydroxylated-diclofop-acid, only one of which was the major metabolite produced by CYP81As. RNA-seq followed by real-time PCR in the crossed progeny of MHR and sensitive lines identified several P450 genes whose overexpressions were associated with MHR. Gene functional characterization revealed that only CYP709C69 conferred diclofop-methyl resistance in rice calli and produced another hydroxylated-diclofop-acid in yeast, reinforcing the relatively low activity of CYP81As to diclofop-methyl. Plants transformed with CYP709C69 had unchanged sensitivity to 46 herbicides except for clomazone, where transgenic plants became more susceptible. The present findings establish a novel concept that simultaneous overexpression of herbicide-metabolizing genes enhances and broadens the profile of metabolic resistance in weeds.

Enhancement of Biocontrol Efficacy of Pichia kudriavzevii Induced by Ca Ascorbate against Botrytis cinerea in Cherry Tomato Fruit and the Possible Mechanisms of Action

Antagonistic yeast is a promising way to control postharvest fruit decay because of its safety and broad-spectrum resistance. However, the biocontrol efficacy of yeast is limited by environmental stress, such as oxidative stress.

A fungal plant pathogen overcomes conserved broad-spectrum disease resistance by rapid gene loss

Hosts and pathogens typically engage in an evolutionary arms race. This also applies to phytopathogenic powdery mildew fungi, which can rapidly overcome plant resistance and perform host jumps. Using experimental evolution, we show that the powdery mildew pathogen Blumeria graminis f.sp. hordei is capable of breaking the agriculturally important broad-spectrum resistance conditioned by barley loss-of-function mlo mutants. Partial mlo virulence is associated with a distinctive pattern of adaptive mutations, including small-sized (8-40 kb) deletions, one of which likely affects spore morphology. The detected mutational spectrum comprises the same loci in at least two independent mlo-virulent isolates, indicating convergent multigenic evolution. This work highlights the dynamic genome evolution of an obligate biotrophic plant pathogen with a transposon-enriched genome.

Optimizing RNAi-Target by Nicotiana benthamiana-Soybean Mosaic Virus System Drives Broad Resistance to Soybean Mosaic Virus in Soybean

Soybean mosaic virus (SMV) is a prevalent pathogen of soybean (Glycine max). Pyramiding multiple SMV-resistance genes into one individual is tedious and difficult, and even if successful, the obtained multiple resistance might be broken by pathogen mutation, while targeting viral genome via host-induced gene silencing (HIGS) has potential to explore broad-spectrum resistance (BSR) to SMV. We identified five conserved target fragments (CTFs) from S1 to S5 using multiple sequence alignment of 30 SMV genome sequences and assembled the corresponding target-inverted-repeat constructs (TIRs) from S1-TIR to S5-TIR. Since the inefficiency of soybean genetic transformation hinders the function verification of batch TIRs in SMV-resistance, the Nicotiana benthamiana-chimeric-SMV and N. benthamiana-pSMV-GUS pathosystems combined with Agrobacterium-mediated transient expression assays were invented and used to test the efficacy of these TIRs. From that, S1-TIR assembled from 462 bp CTF-S1 with 92% conservation rate performed its best on inhibiting SMV multiplication. Accordingly, S1-TIR was transformed into SMV-susceptible soybean NN1138-2, the resistant-healthy transgenic T1-plants were then picked out via detached-leaf inoculation assay with the stock-plants continued for progeny reproduction (T1 dual-utilization). All the four T3 transgenic progenies showed immunity to all the inoculated 11 SMV strains under individual or mixed inoculation, achieving a strong BSR. Thus, optimizing target for HIGS via transient N. benthamiana-chimeric-SMV and N. benthamiana-pSMV-GUS assays is crucial to drive robust resistance to SMV in soybean and the transgenic S1-TIR-lines will be a potential breeding source for SMV control in field.

A giant NLR gene confers broad-spectrum resistance to Phytophthora sojae in soybean

Phytophthora root and stem rot caused by P. sojae is a destructive soybean soil-borne disease found worldwide. Discovery of genes conferring broad-spectrum resistance to the pathogen is a need to prevent the outbreak of the disease. Here, we show that soybean Rps11 is a 27.7-kb nucleotide-binding site-leucine-rich repeat (NBS-LRR or NLR) gene conferring broad-spectrum resistance to the pathogen. Rps11 is located in a genomic region harboring a cluster of large NLR genes of a single origin in soybean, and is derived from rounds of unequal recombination. Such events result in promoter fusion and LRR expansion that may contribute to the broad resistance spectrum. The NLR gene cluster exhibits drastic structural diversification among phylogenetically representative varieties, including gene copy number variation ranging from five to 23 copies, and absence of allelic copies of Rps11 in any of the non-Rps11-donor varieties examined, exemplifying innovative evolution of NLR genes and NLR gene clusters.